ATP Stimulates Mouse Embryonic Stem Cell Proliferation via Protein Kinase C, Pho - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:58 |只看该作者 |正序浏览 |打印

作者：Jung Sun Heo, Ho Jae Han作者单位：Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea   ?) Y- C: ~) L


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      【摘要】
   This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of 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.
      【关键词】 ATP Mitogen-activated protein kinases Phosphatidylinositol -kinase/Akt Protein kinase C Embryonic stem cells/ Q% j/ C8 ]! Y* d
   INTRODUCTION
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ATP is not only a neurotransmitter but also a potent signaling molecule that plays important biological roles in different cell types . 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 . Therefore, this study examined the effect of ATP on mouse ESC proliferation and its related signaling pathways.8 ~2 z; u- n. x/ K8 c
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MATERIALS AND METHODS) q; o2 g3 t( c/ b9 p& E
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Materials1 A+ I( f2 `* ?4 F
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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-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). 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) , PKC , PKC , 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.! Q& d  T' Z" z3 J3 s6 X
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ESC Culture
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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¡ãC in an air atmosphere containing 5% CO2. After 2¨C3 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.
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Alkaline Phosphatase Staining* a5 ^; R9 T% B6 V9 w2 D- S( \& R
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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 ) for 10 minutes at room temperature. After being washed with PBS, the cells were photographed.7 r) G7 b- j! \; T

Thymidine Incorporation- J# S  d  H8 [, X( A% C# E0 f( G
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The 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).
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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.' T4 J5 _, B6 b! }! d, E
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5-Bromo-2'-Deoxyuridine Incorporation. V2 ?4 V# ]2 b2 y
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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%  for 10 minutes at 4¡ãC), 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¡ãC. 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 ¡À SE number of BrdU-positive cells per field of vision was determined. At least 10 fields of vision per coverslip were counted./ t- n2 C% `; l1 {! K# J* T
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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).( K- p% ?, [2 v
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Fluorescence-Activated Cell Sorter Analysis
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Cells were incubated with ATP (10¨C4 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).2 R3 k( Z4 i. u% t5 R9 p! R% N

Measurement of i  \1 t7 f1 `8 @/ k

The changes in i were processed at a single-cell level and are expressed as the relative fluorescence intensity.$ N2 Q* _7 |" H2 [" r$ y0 `

Inositol Phosphate Formation Assay
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The assay was performed using a modification of the method reported by Berridge et al. inositol phosphates (IP1, IP2, and IP3) was eluted with 1 M ammonium formate and 0.1 N formic acid.
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RNA Isolation and Reverse Transcription-Polymerase Chain Reaction1 f( y% I7 i6 c% K

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¡ãC for 5 minutes and 30 cycles at 94¡ãC for 45 seconds, 55¡ãC for 30 seconds, and 72¡ãC for 30 seconds, followed by 5 minutes of extension at 72¡ãC. The primers used were 5'-CGTGAGACTTTGCAGCC-TGA-3' (sense), 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct4 (519 base pair ); 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
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Mouse ESCs were treated with 10¨C4 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¡ãC for reverse transcription; 15 minutes at 95¡ãC for DNA polymerase activation; 15 seconds at 95¡ãC for denaturing; 45 cycles of 15 seconds at 94¡ãC, 30 seconds at 55¡ãC, and 30 seconds 72¡ãC. The data collection was carried out during the extension step (30 seconds at 72¡ãC). 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¡ãC to 99¡ãC at a rate of 1¡ãC per 5 seconds, and the resulting data were analyzed by using the software provided by the manufacturer.
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cAMP Assay
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The mouse ESCs were preincubated with 100 µM 3-isobutyl-1-methylxanthine for 30 minutes at 37¡ãC to inhibit cAMP degradation and incubated with 10¨C4 M ATP for 8 hours at 37¡ãC 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¡ãC. After centrifugation at 900g for 5 minutes, the supernatants were transferred into new tubes and stored at 4¡ãC. The intracellular cAMP levels in the samples were examined using a cAMP assay system kit. The values are expressed as picomoles of cAMP per milligram of protein.
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Preparation of Cytosolic and Total Membrane Fractions9 a" w) l7 v& c& m6 i
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The cytosolic and total membrane fractions were prepared using a slight modification of the method reported by Mackman et al. .
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Western Blot Analysis
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The cells were harvested, washed twice with PBS, and lysed with a buffer (20 mM Tris , 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, v$ |; E! |0 l4 L% e' d. C
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The results are expressed as a mean ¡À 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

RESULTS
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Effect of ATP on Cell Proliferation
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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. 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¨C60 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¨C4 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.0 o; M1 F0 m' P/ r) G! X/ B; l
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The effect of ATP on cell proliferation was examined by treating mouse ESCs with ATP (10¨C4 M) for various time periods (0¨C12 hours) or doses (0¨C5 x 10¨C4 M) for 8 hours. As shown in Figure 2A, the maximum increase in the level of thymidine incorporation (Fig. 3B). In addition, the mouse ESCs expressed the P2X3, P2X4, P2Y1, and P2Y2 receptors but not P2X1 or P2X2 (Fig. 3C).6 m4 P  K; ^% Z. w% V
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Figure 2. Effects of ATP on thymidine for 1 hour. (C): BrdU-positive cells in response to different ATP concentrations (0¨C5 x 10¨C4 M) for 24 hours. The mean ¡À 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¨C4 M) for 24 hour and double-labeled with Oct4 and BrdU antibody. Scale bars = 20 µm. (E): Mouse ESCs were treated with ATP (0¨C5 x 10¨C4 M) for 24 hours, and the number of cells was counted using a hemocytometer. The values represent mean ¡À SE of four independent experiments with triplicate dishes. *, p 2 A  X+ G, _0 H! P: L

Figure 3. Effect of ATP agonists and P2 purinoceptor antagonists on thymidine for 1 hour. The values represent mean ¡À SE of five independent experiments with triplicate dishes. *, p
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Involvement of cAMP/Phospholipase C/PKC in ATP-Induced Cell Proliferation7 Y1 \) `3 m& d
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The effect of SQ 22536 (adenylate cyclase inhibitor; 10¨C6 M), neomycin or U 73122 (phospholipase C  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 ¡À 8% compared with the control) (Fig. 4C). Subsequently, ATP induced the translocation of PKC , , and  from the cytosol to the membrane compartment (Fig. 4D).

Figure 4. Effect of AMP and phospholipase C/PKC inhibitors on ATP-induced 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¨C120 seconds). The values represent mean ¡À SE of four independent experiments with triplicate dishes. *, p ; w1 ^0 Y; k, I0 L7 O; k9 F+ m3 \* f
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Involvement of i in ATP-Induced Cell Proliferation
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The intracellular Ca2  mobility in response to ATP and its agonists was examined to determine whether ESC proliferation involves the ATP-induced increase in thymidine incorporation but BAPTA-AM had no effect (Fig. 5C).- Y1 t$ ~6 Q( u
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Figure 5. Effect of ATP and ATP agonists on 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 ¡À SE of three independent experiments with triplicate dishes. *, p   w- h# B1 v5 |7 U- |/ y, _

Involvement of PI3K/MAPKs in ATP-Induced Cell Proliferation, N* O& E) G* v; u( p6 H7 g
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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 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).$ W2 }/ P- \4 X" x
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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¨C4 M) for various times (0¨C90 minutes), and the phosphorylation of Akt (A) and p44/42 MAPKs (C) was detected. Mouse ESCs were pretreated with either suramin (10¨C5 M) or RB2 (10¨C6 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¨C6 M) or Akt inhibitor (10¨C5 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 ¡À 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¨C6 M), Akt inhibitor (10¨C5 M), or PD 98059 (10¨C6 M) for 30 minutes prior to the ATP treatment for 8 hours and then pulsed with 1 µCi of thymidine for 1 hour. The values represent mean ¡À SE of four independent experiments with triplicate dishes. *, p

Figure 7. Effect of ATP on cell cycle regulators. Mouse ESCs were treated with ATP (10¨C4 M) for various times (0¨C9 hours) (A) or pretreated with suramin (10¨C5 M), RB2 (10¨C6 M), wortmannin (10¨C6 M), or PD 98059 (10¨C6 M) for 30 minutes prior to incubating the cells with ATP (10¨C4 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 ¡À SE of four experiments for each condition determined from densitometry relative to ß-actin. *, p 0 k! ^$ E, c' [0 k& U- m6 o/ U9 q9 N
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DISCUSSION4 L. H  S- A2 Y1 E" Z

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 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 . However, AMP is not associated with the P2R-induced biological effects of the extracellular nucleotides.
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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 .* m: H5 Q) u  J
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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 . However, the contribution of Akt to cell proliferation was suggested to be cell type-specific and stimulus-dependent.
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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 . 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.
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There is evidence showing that extracellular ATP enhances the expression of the cell cycle regulator proteins . 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

The authors indicate no potential conflicts of interest.3 C) H' b, f2 `) a6 o0 n

ACKNOWLEDGMENTS; ^0 S3 I  S! a+ [. k

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.+ G( X( k: V0 H2 Z0 I
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