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EMBRYONIC STEM CELLS

High-Glucose-Induced Prostaglandin E₂ and Peroxisome Proliferator-Activated Receptor Promote Mouse Embryonic Stem Cell Proliferation

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

Key Words. High glucose • Embryonic stem cells • Peroxisome proliferator-activated receptor • Prostaglandin E₂

Correspondence: 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 on September 20, 2007; accepted for publication on December 14, 2007.

First published online in STEM CELLS EXPRESS  December 20, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Peroxisome proliferator-activated receptor is a nuclear receptor that has been implicated in blastocyst implantation, cell cycle, and pathogenesis of diabetes. However, the signal cascades underlying this effect are largely unknown in embryo stem cells. This study examined whether or not there is an association between the reactive oxygen species-mediated prostaglandin E₂ (PGE₂)/peroxisome proliferator-activated receptor (PPAR) and the growth response to high glucose levels in mouse ESCs. A high concentration of glucose (25 mM) significantly increased the level of [³H]thymidine incorporation, the level of 5-bromo-2'-deoxyuridine incorporation, and the number of cells. Moreover, 25 mM glucose increased the intracellular reactive oxygen species, phosphorylation of the cytosolic phospholipase A₂ (cPLA₂), and the release of [³H]arachidonic acid ([³H]AA). In addition, 25 mM glucose also increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of PGE₂. Subsequently, high glucose-induced PGE₂ stimulated PPAR expression directly or through Akt phosphorylation indirectly through the E type prostaglandin receptor receptors. The PPAR antagonist inhibited the 25 mM glucose-induced DNA synthesis. Moreover, transfection with a pool of PPAR-specific small interfering RNA inhibited the 25 mM glucose-induced DNA synthesis and G1/S phase progression. Twenty-five millimolar glucose also increased the level of the cell cycle regulatory proteins (cyclin E/cyclin-dependent kinase [CDK] 2 and cyclin D1/CDK 4) and decreased p21^WAF1/Cip1 and p27^Kip1, which were blocked by the inhibition of the cPLA₂, COX-2, or PPAR pathways. In conclusion, high glucose promotes mouse ESC growth in part through the cPLA₂-mediated PGE₂ synthesis and in part through PPAR pathways.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Glucose provides more than just a source of combustible energy. It also produces a wide variety of cellular signals. In this way, glucose provides a mechanism through which the broadly defined outside "environment" can communicate directly with the specific cell/tissues in response to either glucose or excess glucose. These cellular responses to glucose uptake can also be a source of embryopathy, including the relationship between glucose uptake and diabetes [1]. There are wider implications regarding the effect of glucose concentration on the differentiation of ESCs into other cell types, because early human and mouse embryo development in vitro has been shown to be enhanced in a medium lacking glucose [2, 3]. However, the protocols for examining ESC differentiation mainly used media containing high glucose concentrations [4]; their use is presumably based on the use of high-glucose media for maintaining the ESCs. Therefore, there is some debate as to the necessity of using media with a high glucose level for an ESC culture.

Recent work has established peroxisome proliferator-activated receptors (PPARs) as one mechanism through which glucose-driven transcriptional regulation can occur [5]. Three PPAR isotypes, PPAR, PPARβ/, and PPAR, have been identified [6]. PPAR is expressed mainly in the liver, heart, kidney, brown adipose tissue, and stomach mucosa [7, 8]. PPAR is found primarily in the adipose tissue [9]; PPARβ/ is the most ubiquitously expressed, even though its physiological and pathophysiological roles are less clear, particularly in human tissue [10]. PPAR is the only PPAR isoform expressed during rat early embryo organogenesis [11]. In addition, PPAR has been linked to the proliferation of colon cancer, preadipocyte proliferation, and embryo implantation. Therefore, we hypothesize that PPAR plays an important role in the proliferation of ESCs. A previous study demonstrated that both prostaglandin E₂ (PGE₂) and arachidonic acid (AA) supplementation show in vivo and in vitro protection against diabetic malformations [12]. Moreover, in various cell types, PGE₂ production was increased, whereas it was decreased by cyclooxygenase-2 (COX-2) inhibitor, when cells were exposed to high concentration of glucose [13]. Moreover, the high-glucose-induced increase in PGE₂ production requires activation of protein kinase C (PKC) and mitogen-activated protein kinase pathways [14], as well as PKC-induced phospholipase A₂ (PLA₂) activation and the release of arachidonic acid [15]. Although several studies have shown that PPAR plays an important role in the lipid metabolism and that specific agonists might be effective in improving the metabolic syndrome, the physiological implications of the endogenous AA metabolism in the activation of PPAR in ESCs and the functions of PPAR in mouse ESC proliferation exposed to high glucose levels are largely unknown.

ESCs are pluripotent cell lines that are derived from the blastocyst stage of early mammalian embryos. These unique cells are characterized by their capacity for prolonged undifferentiated proliferation in tissue culture, as well as by their ability to maintain their potential to differentiate into derivatives of all three germ layers [16]. Therefore, mouse ESCs might be regarded as a versatile biological system, and their use has led to major advances in cell and developmental biology. The growth of mouse ESCs in culture is likely to require the increased uptake of glucose and other substrates [1]. Therefore, mouse ESCs might be useful for examining the effect of high glucose levels on the proliferation of the inner cell mass in early embryos. This study was designed to determine whether there is an association between the PGE₂/PPAR mediated by reactive oxygen species and the growth response to high glucose in mouse ESCs.

	MATERIALS AND METHODS

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Materials
The mouse ESC line was obtained from 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 D-glucose, mannitol, LY294002, N-acetylcysteine (NAC), ascorbic acid, arachidonyltrifluoro-methyl ketone, mepacrine, indomethacin, PGE₂, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM, and β-actin were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). The Akt inhibitor GW9662 and L-165041 were purchased from Calbiochem (La Jolla, CA, http://www.emdbiosciences.com). [³H]Thymidine was purchased from NEN (Cambrex). Fluo 3-AM was supplied by Molecular Probes (Eugene, OR, http://probes.invitrogen.com). The phospho-cytosolic phospholipase A₂ (phospho-cPLA₂), total cPLA₂, phospho-Akt (Thr³⁰⁸, Ser⁴⁷³), total Akt, PPAR, cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Goat anti-rabbit IgG was acquired from Jackson Immunoresearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com). Liquiscint was obtained from National Diagnostics (Parsippany, NY, https://www.nationaldiagnostics.com). All other reagents were of the highest purity commercially available.

ESC Culture
The mouse ESCs were cultured in Dulbecco's modified Eagle's medium (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 β-mercaptoethanol, 5 ng/ml mouse leukemia inhibitory factor (LIF), and 15% fetal bovine serum (FBS) without a feeder layer and cultured for 5 days in standard medium plus LIF. The cells were grown on gelatinized 12-well plates or a 60-mm culture dish in an incubator maintained at 37°C in an atmosphere of 5% CO₂ in air. The medium was changed to serum-free DMEM with LIF before experiments. After that, the cells were washed twice with phosphate-buffered saline (PBS) and then maintained in a serum-free DMEM (5 mM glucose) including all supplements and indicated agents.

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 using an alkaline phosphatase substrate solution (200 µg/ml naphthol AS-MX phosphate [3-Hydroxy-2-naphthoic Acid 2, 4-Dimethylanilide 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.

Immunofluorescence Staining with SSEA-1
The cells were fixed and treated with the monoclonal antibody against mouse SSEA-1 (1:50; Santa Cruz Biotechnology) and incubated for 30 minutes with the FITC-conjugated secondary antibodies raised in rabbit against mouse IgM (1:100). The fluorescence images were visualized using a fluorescence microscope (Fluoview 300; Olympus, Tokyo, http://www.olympus-global.com).

[³H]Thymidine Incorporation
The [³H]thymidine incorporation experiments were carried out using the methodology reported by Brett et al. [17]. Zhang et al. [18] reported that most ESCs could be arrested in the G0/G1 phase using a serum deprivation culture. Furthermore, the synchronized ESCs could successfully reenter a normal cell cycle after being resupplied with the serum. The cells were washed twice with PBS and incubated with fresh serum-free DMEM (5 mM glucose) 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, Little Chalfont, U.K., http://www.amersham.com) was added to the cultures. The incubation with [³H]thymidine was continued for an additional 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 0.2 N NaOH for 12 hours at 23°C. Aliquots were removed to determine the level of radioactivity using a liquid scintillation counter (LS 6,500; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). All values are reported as the mean (± SE) of triplicate experiments. The values were converted from absolute counts to a percentage of the control to allow for a comparison between experiments.

5-Bromo-2'-Deoxyuridine Incorporation
The level of 5-bromo-2'-deoxyuridine (BrdU) (a thymidine analog) incorporation was measured to determine the level of DNA synthesis. The ESCs were serum-starved for 24 hours before being stimulated with 25 mM glucose. The ESCs were then treated with 25 mM glucose for 12 hours. Fifteen micromolar BrdU was added during the final 16 hours of incubation. 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 washed and then incubated for 15 minutes with 0.1 M sodium tetraborate. 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 mounting medium (Dako, Glostrup, Denmark, http://www.dako.com) using gelvatol and examined by optical microscopy (Fluoview 300; Olympus). The mean ± SE number of BrdU-positive cells per field of vision was determined. Ten fields of vision per coverslip were counted.

For the double-labeling experiments, the cells were fixed in acidified alcohol and processed for Oct-4 staining, which was followed by BrdU staining. The fixed cells were incubated with the rabbit anti-Oct-4 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 and neutralization with 0.1 M sodium tetraborate and then with Alexa Fluor 488-conjugated mouse anti-BrdU mAb for 1 hour at room temperature. After being washed with PBS, the BrdU/Oct-4-stained cells were examined by confocal microscopy (Fluoview 300; Olympus).

Cell Proliferation Assay
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.

H₂O₂ Release
The H₂O₂ levels were determined using a modification of the method reported by Zhou et al. [19]. The cells were washed twice with ice-cold PBS, harvested by microcentrifugation, and resuspended in Krebs-Ringer phosphate solution (KRPG) (145 mM NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mM CaCl₂, 1.22 mM MgSO₄, 5.5 mM glucose [pH 7.35]). One hundred microliters of the reaction mixture (50 µM Amplex Red reagent containing 0.1 U/ml horseradish peroxidase in KRPG) was added to each microplate well that had been prewarmed to 37°C for 10 minutes. The reaction was started by adding 20 µl of KRPG to the resuspended cells. The fluorescence readings became stable within 30 minutes of starting the reaction, and the absorbance at 560 nm was then measured using a fluorescence microplate reader (Multiskan; Thermo Labsystems Inc., Franklin, MA, http://www.thermo.com).

Measurement of Lipid Peroxides
The lipid peroxide (LPO) levels in the mouse ESCs were determined by measuring the malondealdehyde content using the method reported by Ohkawa et al. [20]. One hundred microliters of the sonicated cells was mixed with 100 µl of an 8% sodium dodecyl sulfate (SDS) solution, 200 µl of a 0.8% 2-thiobarbituric acid solution, and 200 µl of a 20% acetic acid solution. The mixture was heated to 95°C for 60 minutes. After incubation, the mixture was cooled in ice-cold water. The nonspecific red pigment was extracted by adding 1 ml of an n-butanol-pyridine mixture (15:1 [vol/vol]) and centrifuging the sample at 1,550g for 10 minutes. The organic supernatant was measured by spectrofluorometry at emission and excitation wavelengths of 553 and 515 nm, respectively. 1,1,3,3-Tetraethoxypropane was used as the standard, and the LPO values of the samples are expressed as nmol/mg protein.

Assay of Cellular Reactive Oxygen Species
The intracellular production of reactive oxygen species (ROS) was measured using confocal microscopy according to the method reported by Lee et al. [21]. Generation of ROS was assessed using the fluorescence indicator 5-(and-6)-chloromethyl-2,7-dichlorodihydro-fluorescein diacetate (CM-H₂DCF-DA) (Molecular Probes), which becomes highly fluorescent upon oxidation by intracellular H₂O₂ [22, 23]. To confirm involvement of ROS in 25 mM glucose-induced cell proliferation, mouse ESCs were treated with NAC (10^–5 M) before being treated with either 25 mM glucose or H₂O₂ (5 µM) for 1 hour. The cells were washed with Dulbecco's PBS and incubated for 15 minutes in Krebs-Ringer solution containing 5 µM CM-H2DCF-DA. The ROS generation was detected (excitation, 488 nm; emission, 515–540 nm) using a fluorescent microscope (Fluoview 300; Olympus).

Fluorescence-Activated Cell Sorting Analysis
Cells were incubated with 25 mM glucose for 24 hours, and the cells were dissociated in trypsin/EDTA, pelleted by centrifugation, and resuspended at approximately 10⁶ cells per milliliter in PBS containing 0.1% BSA. The cells were fixed in 70% ice-cold ethanol, followed by incubation in a freshly prepared nucleus-staining buffer (250 µg/ml propidium iodide [PI] and 100 µg/ml RNase) for 30 minutes at 37°C. The cell cycle histograms were generated after analyzing the PI-stained cells by fluorescence-activated cell sorting (Beckman Coulter). At least 10⁴ events per sample were recorded. The samples were analyzed using CXP software (Beckman Coulter).

AA Release
To quantitate AA release by modification of the method of Xing et al. [24], confluent mouse ESCs were incubated for 24 hours (0.5 µCi/ml [³H]AA in serum-free DMEM including LIF). The monolayers were then washed and incubated for 1 hour (37°C; DMEM). At the end of the incubation, the medium was transferred to ice-cold tubes containing 55 mM EGTA and 5 mM EDTA and centrifuged (12,000g), and soluble material was counted in a liquid scintillation counter. Both the [³H]AA released and cell-associated [³H]AA were standardized with respect to protein. Then, released [³H]AA was compared percentagewise to cell-associated [³H]AA (present at the beginning of the incubation).

PPAR Small Interfering RNA
The cells were grown in each dish until they reached 75% confluence. They were then transfected for 24 hours with either a SMARTpool of the small interfering RNAs specific to PPAR (200 pmol/l) or a nontargeting small interfering RNA (as negative control; 200 pmol/l; Dharmacon, Inc., Lafayette, CO, http://www.dharmacon.com) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Preparation of Cytosolic and Total Membrane Fractions
The preparation of the cytosolic and total membrane fractions was performed using a modification of the method reported by Mackman et al. [25]. 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 [pH 7.5]). The resuspended cells were then lysed mechanically on ice by trituration with a 21.1-gauge needle. The lysates were first centrifuged at 1,000g for 10 minutes at 4°C. The supernatants were centrifuged at 100,000g for 1 hour at 4°C to prepare the cytosolic and total particulate fractions. The supernatants (cytosolic fraction) were then precipitated with 5 volumes of acetone, incubated for 5 minutes on ice, 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, which contained 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 [26].

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from mouse ESCs using STAT-60 monophasic solution of phenol and guanidine isothiocyanate from Tel-Test (Friendswood, TX, http://www.isotexdiagnostics.com). Reverse transcription was conducted with 3 µg of RNA using a reverse transcription system kit (AccuPower RT PreMix, Bioneer, Daejeon, Republic of Korea, http://www.bioneer.com) with oligo(dT)₁₈ primers. After that, 5 µl of reverse transcription (RT) products was amplified with a polymerase chain reaction (PCR) kit (AccuPower PCR PreMix, Korea), followed by denaturation at 94°C for 5 minutes and 30 cycles at 94°C for 15 seconds, 55°C for 1 minute, and 72°C for 45 seconds, followed by a 5-minute extension at 72°C. Amplifications of Oct4, FOXD3, SOX2, EP (1, 2, 3, 4), and PPAR (, , ) cDNAs were performed in mouse ESCs using primers described in Table 1. PCR of β-actin was also performed as control for quantity of RNA.

PGE₂ Assay
Mouse ESCs plated on 60-mm culture plates were grown in an FBS-free medium for 24 hours and divided into groups according to the experimental protocol. The PGE₂ concentration in the culture medium was measured using an enzyme-linked immunosorbent assay with a PGE₂ High Sensitivity Immunoassay kit (R&D Systems Inc., Minneapolis, http://www.rndsystems.com).

Western Blot Analysis
The cell homogenates (containing 20 µg of protein) were separated by electrophoresis through 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The blots were then washed with H₂O, blocked for 1 hour with 5% skim milk powder in Tris-buffered saline/Tween 20 (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween 20), and incubated with the appropriate primary antibody at the dilutions recommended by the supplier. The nitrocellulose membrane was washed, and the primary antibodies were detected with either goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase. The bands were visualized by enhanced chemiluminescence (Amersham Biosciences U.K., Little Chalfont, Buckinghamshire, U.K., http://www.amershambiosciences.com).

Statistical Analysis
The results are expressed as the mean ± SE The difference between two mean values was analyzed using a Student t test. A p value <.05 was considered significant.

	RESULTS

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Effect of High Glucose on Mouse ESC 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 Oct4, FOXD3, and SOX2 expression. The mouse ESCs expressed Oct4, FOXD3, and SOX2 mRNA in both the presence and the absence of 25 mM glucose (Fig. 1A). Moreover, the cells in the presence of 25 mM glucose expressed an amount of the Oct4 protein equivalent to that of the control (Fig. 1B) and maintained the alkaline phosphatase enzyme activity (Fig. 1C). In addition, expression of the SSEA-1 protein was identified by immunostaining (Fig. 1D). Therefore, these results indicate that mouse ESCs maintain an undifferentiated state under these conditions.

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of high glucose levels on the characterization of mouse embryonic stem (ES) cells. (A): Oct4, FOXD 3, SOX 2, and β-actin mRNA expression levels in the presence or absence of 25 mM glucose. (B): Oct4 and β-actin protein expression levels in the presence or absence of 25 mM glucose. The bands represent 50–60 kDa of Oct4 and 41 kDa of β-actin. (C): The alkaline phosphatase enzyme activity in the cells was measured in the presence or absence of 25 mM glucose, as described in Materials and Methods. (D): Immunofluorescence staining of mouse ES cells with the mouse stage-specific embryonic antigen-1-specific antibody.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of high glucose levels on mouse ESCs proliferation. (A): The mouse ESCs were incubated with various concentrations of glucose or mannitol (5–50 mM) and pulsed with 1 µCi of [3H]thymidine for 1 hr. *, p < .05 versus 5 mM glucose. (B): BrdU-positive cells in response to different glucose concentrations (5 or 25 mM) for 12 hr. The number of BrdU-positive cells per field of vision was determined. At least 10 fields of vision per coverslip were counted. *, p < .05 versus 5 mM glucose. (C): The mouse ESCs were incubated with 25 mM glucose for 24 hr and double-labeled with the Oct4 and BrdU antibody. (D): The mouse ESCs were treated with glucose (5 or 25 mM) for 12 hr, and the cells were counted using a hemocytometer. The values represent the mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; hr, hours.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of high glucose levels on the cellular level of reactive oxygen species (ROS). (A, B): H2O2 (A) and lipid peroxide (B) formation was measured after the mouse ESCs had been treated with 25 mM glucose. The values represent the mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose. (C): Dichlorofluorescein-sensitive cellular ROS was measured by confocal microscopy. (D): Mouse ESCs were incubated with 25 mM glucose for 0–120 min and harvested. The total protein was extracted and blotted with the phospho- or total cPLA2 antibodies. The example shown is a representative of four independent experiments. *, p < .05 versus 5 mM glucose. (E): Mouse ESCs were pretreated with NAC and ascorbic acid for 30 min before being treated with 25 mM glucose for 30 min. The total protein was extracted and blotted with phospho- or total cPLA2 antibodies. The example shown is a representative of four independent experiments. The graphs (D, E) denote the mean ± SE of four experiments for each condition determined from densitometry relative to each total cPLA2. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (F): NAC (10–5 M), ascorbic acid (10–3 M), AACOCF3 (10–6 M), and mepacrine (10–6 M) significantly blocked the 25 mM glucose-induced increase in [3H]AA release. The values represent the mean ± SE of three independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (G): Mouse embryonic stem cells were pretreated with NAC, ascorbic acid, AACOCF3, and mepacrine for 30 min before being treated with 25 mM glucose for 12 hours. The cells were then pulsed with 1 µCi of [3H]thymidine for 1 hour. The values represent the mean ± SE of five independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. Abbreviations: AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; min, minutes; NAC, N-acetylcysteine.

View larger version (33K): [in this window] [in a new window]   Figure 4. Effect of high glucose concentrations on the level of cyclooxygenase/PGE2 activation. (A): Mouse ESCs were incubated with 25 mM glucose for 0–24 hr and then harvested. (B): Mouse ESCs were pretreated with AACOCF3 and mepacrine for 30 minutes before being treated with 25 mM glucose for 12 hr. (C): Effect of AACOCF3 and indomethacin on 25 mM glucose-induced PGE2 production. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (D): Mouse ESCs were treated with 25 mM glucose for 12 hr, and the level of EP1, EP2, EP3, and EP4 receptor gene expression was then analyzed by reverse transcription-polymerase chain reaction (RT-PCR) (Da) and real-time RT-PCR (Db). Each example shown is representative of three independent experiments. *, p < .05 versus 5 mM glucose. (E): Mouse ESCs were treated with either 25 mM glucose or PGE2 for 12 hr. The cells were then pulsed with 1 µCi of [3H]thymidine for 1 hr. *, p < .05 versus 5 mM glucose alone. (F): Mouse ESCs were treated with 25 mM glucose and PGE2 for 30 minutes. The cells were then harvested. The total protein was extracted and blotted with phospho-Akt (Thr308, Ser473) antibodies or total Akt. The example shown is a representative of four independent experiments. Abbreviations: hr, hours; PGE2, prostaglandin E2.

Effect of High Glucose Levels on PPAR Activation

View larger version (30K): [in this window] [in a new window]   Figure 5. Effect of high glucose levels on PPAR gene expression. (A): Mouse ESCs were treated with 25 mM glucose for 12 hr, and the PPAR, , and gene expression levels were then analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The PPAR , , and gene expression levels were then analyzed using RT-PCR (Aa) and real-time RT-PCR (Ab). Each example shown is representative of three independent experiments. *, p < .05 versus 5 mM glucose. (B): Mouse ESCs were incubated with 25 mM glucose for 0–24 hr and then harvested. The example shown is a representative of four independent experiments. The graph denotes the mean ± SE of four experiments for each condition determined from densitometry relative to each β-actin. *, p < .05 versus 0 minutes. (C): Mouse ESCs were pretreated with NAC, AACOCF3, and indomethacin for 30 minutes before being treated with 25 mM glucose for 12 hr. The example shown is a representative of four independent experiments. The graph denotes the mean ± SE of four experiments for each condition determined from densitometry relative to each β-actin. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (D): Mouse ESCs were transfected for 24 hr with either a SMARTpool of PPAR siRNAs (200 pmol/l) or a nontargeting control siRNA (200 pmol/l) using Lipofectamine 2000 prior to 25 mM glucose treatment for 24 hr. The cells were then pulsed with 1 µCi of [3H]thymidine for 1 hr. The values represent the mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (E): Mouse ESCs were pretreated with L-165041 (PPAR agonist: 10–6 M), and GW9662 (PPAR antagonist: 10–6 M) for 30 minutes prior being treated with 25 mM glucose for 12 hr. The cells were then pulsed with 1 µCi of [3H]thymidine for 1 hr. The values represent the mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. (F): The mouse ESCs were pretreated with LY294002 and the Akt inhibitor for 30 minutes before being treated with 25 mM glucose for 12 hr. The graph denotes the mean ± SE of four experiments for each condition determined from densitometry relative to each β-actin. *, p < .05 versus 5 mM glucose; **, p < .05 versus 25 mM glucose alone. Abbreviations: hr, hours; NAC, N-acetylcysteine; PPAR, peroxisome proliferator-activated receptor; siRNA, small interfering RNA.

Effect of High-Glucose-Induced PPAR on Cell Cycle Regulatory Proteins

View larger version (30K): [in this window] [in a new window]   Figure 6. Effect of PPAR on the high-glucose-induced increase in the level of the cell cycle regulatory proteins. (A): Mouse ESCs were pretreated with AACOCF3, indomethacin, and GW9662 for 30 minutes before being treated with 25 mM glucose for 12 hours. The total protein was extracted and blotted with the cyclin D1, cyclin E, CDK 2, CDK 4, p21WAF1/Cip1, and p27Kip1 antibodies. Each example shown is a representative of four independent experiments. (B): Mouse ESCs were transfected for 24 hours with either a SMARTpool of PPAR siRNAs (200 pmol/l) or a nontargeting control siRNA (200 pmol/l) using Lipofectamine 2000 prior to 25 mM glucose treatment for 24 hours. (C): Effect of 25 mM glucose on cell cycle progression. Representative fluorescence-activated cell sorting data (Ca) and corresponding histograms (Cb) for the mouse ESCs, which were pretreated with GW9662 before the 24-hour 25 mM glucose treatment. The cells were washed with phosphate-buffered saline, fixed, stained, and analyzed by flow cytometry. The gates were configured manually to determine the percentage of cells in the G1, S, and G2 phases on the basis of the DNA content. The values represent the mean ± SE of four independent experiments. Abbreviations: CDK, cyclin-dependent kinase; PPAR, peroxisome proliferator-activated receptor; siRNA, small interfering RNA.

	Discussion

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The present result showed that the ROS level in mouse ESCs exposed to high D-glucose concentrations for 24 hours was higher than that in the control. This suggests that oxidative stress is a key factor in the etiology of the associated proliferative anomalies in cells cultured in high D-glucose concentrations. The origin of this increased ROS generation by high D-glucose is unclear, but a substrate overload in the mitochondrial electron transport chain might induce excessive generation of ROS [29]. Moreover, it has been reported that treatment of the cells with phorbol 12-myristate 13-acetate, a PKC activator, increased ROS [30]. Therefore, high glucose can induce cellular ROS through mitochondrial metabolism and intracellular signaling molecule, indicating PKC-dependent activation of NADPH oxidase [7, 8]. It was also observed that treating mouse ESCs with high glucose levels leads to the activation of cPLA₂ and AA release. Upon activation through phosphorylation and intracellular calcium influx, cPLA₂ is translocated to cellular membranes, releasing AA from membrane phospholipids [31]. Recent studies have shown that ROS enhance AA release and the subsequent AA metabolism in macrophages [32]. In the present study, we provide the first direct evidence for an involvement of COX-2 in the stimulation of mouse ESC proliferation by high glucose. The elevation of glucose significantly increased the expression of COX-2 protein levels. In the present study, the concentration of PGE₂ generated by the AA cascade through COX increased proliferation of mouse ESCs obtained under high glucose level. Previous work has shown that embryos from streptozotocin (STZ)-induced diabetic rats have a diminished PGE₂ content, although they can produce PGE₂ in large amounts [33]. It is interesting to speculate that AA might be depleted if PGE₂ generation and release is increased in the diabetic embryo to maintain intracellular PGE₂ levels [34]. Most interestingly, glucose-induced COX-2 upregulation was associated with a PPAR [35]. The mechanisms by which high glucose increases the expression of PPAR mRNA remain unclear, and further investigation is needed.

In the present study, we demonstrated a significant increase in PPAR expression and a slight upregulation of PPAR in response to high glucose, whereas PPAR was downregulated. Previous studies demonstrated that placental PPAR expression was decreased under mild hyperglycemia in gestational diabetic women or in STZ-induced diabetic rats [36, 37] Thus, we hypothesize that overexpression of PPAR in mouse ESCs is a sufficient condition to increase cell proliferation. In this study, we provided the first evidence for a role of PPAR in the regulation of high-glucose-induced mouse ESC proliferation. We reproduced this observation using a PPAR selective agonist, L-165041, to stimulate the growth of mouse ESCs and used a siRNA system to validate PPAR as the mediator of these effects. The present result is consistent with a recent report that PPAR promotes postconfluent cell proliferation in 3T3 fibroblasts [38]. The physiological and pharmacological roles of PPAR are just beginning to emerge. It has recently become clear that PPAR has a function in epithelial tissues, but inconsistent reports leave the situation controversial. Indeed, some reports suggest that ligand activation of PPAR potentiates cell growth [39], whereas other reports suggest that ligand activation of PPAR attenuates cell growth [40]. More recently, it was suggested that ligand activation of PPAR induces expression of COX-2 [39], which could theoretically promote cell growth and inhibit apoptosis through mechanisms that involve the production of prostaglandins. These data raise the possibility of a model in which impaired activation of PPAR may alter the lipid signaling required for normal self-renewal of ESCs, and they point to PPAR as a putative target in maintenance of mouse ESCs stemness.

As PGE₂ exerts its bioactivity through four different G-protein-coupled EP receptors (EP1, EP2, EP3, and EP4), it was important to evaluate their expression in mouse ESCs. Although the mRNAs for all of four EP receptor subtypes were detected in mouse ESCs, the EP1 receptor mRNA is the most abundant form [41]. The present study also showed that the treatment of high glucose or PGE₂ increased the mRNA level of EP1 receptor. This is supported by a previous study showing that PGE₂ increased the mRNA level of EP1 receptor without affecting the levels of mRNA for other EP receptor subtypes [42]. Another report demonstrated that in the context of high-glucose-induced DNA synthesis in mesangial cells, high glucose stimulated the overproduction of PGE₂ and the augmentation of EP1 function [43, 44]. Our findings in this study provide the first evidence for the expression profiles of EP receptors in mouse ESCs and depict a pivotal role of the EP1 receptor in mouse ESC proliferation. The generation of phosphatidylinositol 3,4,5-triphosphate on the inner layer of the plasma membrane following PI3K activation recruits Akt by direct interaction with its pH domain. Phosphorylated Akt dissociates from the membrane and enters the cytoplasm and nucleus, where it phosphorylates several key proteins mediating cellular effects such as stimulation of cell cycle progress [41]. Various other studies using fibroblasts and other cell types have also shown that PGE₂ mediates its regulatory effects on various downstream targets via PI3K and Akt signaling pathways [45, 46]. Our present data demonstrate a direct effect of PGE₂ on Akt phosphorylation via EP receptor in mouse ESCs.

The cell cycle is dysregulated under high-glucose conditions, and G1 phase cell cycle progression is believed to be responsible for the glucose-induced mouse ESC proliferation [27]. There is growing evidence that specific CDK inhibitors p21^WAF1/Cip1 and p27^Kip1 are critically involved in the G1 phase cell cycle progression in mouse ESCs exposed to a high glucose level [47]. Based upon these and previous results, it is hypothesized that PPAR activation increases the level of binding of the Adenovirus E2 promoter-binding factor/DNA-binding heterodimerization partner protein heterodimers to their target genes, and the PPAR ligands might increase the level of pRb phosphorylation and accelerate G1/S phase transition in mouse ESCs. In addition, PPAR upregulates cyclin/CDK and mediates G1 cell cycle progression. Therefore, high-glucose treatment results in G1 cell cycle progression and increased proliferation. Figure 7 shows a hypothetical model of the signaling mechanisms involved in mediating the high-glucose-induced proliferation of mouse ESCs. Consistent with present results, overexpression of PPAR in vascular smooth muscle cell increased cell proliferation by increasing the cyclin A and CDK 2, as well as decreasing p57^Kip2 [39]. Although the proliferative properties as a result of inducing G1 cell cycle progression are associated with the activation of PPAR activation by high glucose in various cell types, it appears that the effect of a high glucose level on cell cycle and proliferation depends on the cell cycle or conditions. The present study can be regarded as an extension of studies aimed at elucidating the functional role of a high concentration of glucose in mouse ESC expansion and demonstrating the value and the advantages of this factor in the ESC culture system. On the basis of these results, we suggest that the series of high-glucose experiments provide a fascinating insight into the role of a high concentration of glucose in the expansion of ESCs and the establishment of a stable culture system for ESCs associated with maintenance of self-renewal. In conclusion, these results demonstrate that mouse ESC growth was enhanced by high-glucose-induced PPAR, which is mediated, at least in part, through the induction of COX-2 expression and PGE₂ production.

View larger version (39K): [in this window] [in a new window]   Figure 7. The hypothesized model for the signal pathways involved in high-glucose-induced mouse ESC proliferation. High glucose increased PGE2 synthesis, which is controlled by the coupled activation of cPLA2/AA and COX-2 via ROS. After PGE2 is released into the extracellular space, it binds to the membrane coupled EP receptors. This effect is mediated, at least in part, by the activation of PI3K/Akt. Finally, these molecules may induce PPAR, which increases cell cycle regulatory protein expression levels. Abbreviations: AA, arachidonic acid; CDK, cyclin-dependent kinase; COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; NAC, N-acetylcysteine; PGE2, prostaglandin E2; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... 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 of Korea. We acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.

	REFERENCES

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1. Heilig C, Brosius F, Siu B et al. Implications of glucose transporter protein type 1 (GLUT1)-haplodeficiency in embryonic stem cells for their survival in response to hypoxic stress. Am J Pathol 2003;163:1873–1885.[Abstract/Free Full Text]

2. Conaghan J, Handyside AH, Winston RM et al. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Fertil 1993;99:87–95.[Abstract/Free Full Text]

3. Quinn P. Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J Assist Reprod Genet 1995;12:97–105.[Medline]

4. Chang KH, Zandstra PW. Quantitative screening of embryonic stem cell differentiation: Endoderm formation as a model. Biotechnol Bioeng 2004;88:287–298.[CrossRef][Medline]

5. Brown JD, Plutzky J. Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets. Circulation 2007;115:518–533.[Abstract/Free Full Text]

6. Willson TM, Brown PJ, Sternbach DD et al. The PPARs: From orphan receptors to drug discovery. J Med Chem 2000;43:527–550.[CrossRef][Medline]

7. Sher T, Yi HF, McBride OW et al. cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry 1993;32:5598–5604.[CrossRef][Medline]

8. Auboeuf D, Rieusset J, Fajas L et al. Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: No alteration in adipose tissue of obese and NIDDM patients. Diabetes 1997;46:1319–1327.[Abstract]

9. Chawla A, Schwarz EJ, Dimaculangan DD et al. Peroxisome proliferator-activated receptor (PPAR) : Adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 1994;135:798–800.[Abstract]

10. Han S, Ritzenthaler JD, Wingerd B et al. Activation of peroxisome proliferator-activated receptor β/ (PPARβ/) increases the expression of prostaglandin E₂ receptor subtype EP4. The roles of phosphatidylinositol 3-kinase and CCAAT/enhancer-binding protein β. J Biol Chem 2005;280:33240–33249.[Abstract/Free Full Text]

11. Braissant O, Wahli W. Differential expression of peroxisome proliferator-activated receptor-,β, during rat embryonic development. Endocrinology 1998;139:2748–2754.[Abstract/Free Full Text]

12. Reece EA, Wu YK, Wiznitzer A et al. Dietary polyunsaturated fatty acid prevents malformations in offspring of diabetic rats. Am J Obstet Gynecol 1996;175:818–823.[CrossRef][Medline]

13. Liu H, Peng Y, Liu F et al. A selective cyclooxygenase-2 inhibitor decreases transforming growth factor-β1 synthesis and matrix production in human peritoneal mesothelial cells. Cell Biol Int 2007;31:508–515.[CrossRef][Medline]

14. Sitter T, Haslinger B, Mandl S et al. High glucose increases prostaglandin E₂ synthesis in human peritoneal mesothelial cells: Role of hyperosmolarity. J Am Soc Nephrol 1998;9:2005–2012.[Abstract]

15. Williams B, Schrier RW. Glucose-induced protein kinase C activity regulates arachidonic acid release and eicosanoid production by cultured glomerular mesangial cells. J Clin Invest 1993;92:2889–2896.[Medline]

16. Chambers I. The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells 2004;6:386–391.[CrossRef][Medline]

17. Brett CM, Washington CB, Ott RJ et al. Interaction of nucleoside analogues with the sodium-nucleoside transport system in brush border membrane vesicles from human kidney. Pharm Res 1993;10:423–426.[CrossRef][Medline]

18. Zhang E, Li X, Zhang S et al. Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro. Biochem Biophys Res Commun 2005;333:1171–1177.[CrossRef][Medline]

19. Zhou M, Diwu Z, Panchuk-Voloshina N et al. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 1997;253:162–168.[CrossRef][Medline]

20. Ohkawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–358.[CrossRef][Medline]

21. Lee HB, Yu MR, Song JS et al. Reactive oxygen species amplify protein kinase C signaling in high glucose-incuced fibronectin expression by human peritoneal mesothelial cells. Kidney Int 2004;65:1170–1179.[CrossRef][Medline]

22. Labieniec M, Gabryelak T. Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H₂O₂ or Cu²⁺ ions. Toxicol In Vitro 2007;21:146–156.[CrossRef][Medline]

23. Afzal M, Matsugo S, Sasai M et al. Method to overcome photoreaction, a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species. Biochem Biophys Res Commun 2003;304:619–624.[CrossRef][Medline]

24. Xing M, Tao L, Insel PA. Role of extracellular signal-regulated kinase and PKC in cytosolic PLA₂ activation by bradykinin in MDCK-D1 cells. Am J Physiol 1997;272:C1380–C1387.[Medline]

25. Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med 1991;174:1517–1526.[Abstract/Free Full Text]

26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.[CrossRef][Medline]

27. Kim YH, Heo JS, Han HJ. High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways. J Cell Physiol 2006;209:94–102.[CrossRef][Medline]

28. Sugimoto R, Enjoji M, Kohjima M et al. High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase. Liver Int 2005;25:1018–1026.[CrossRef][Medline]

29. Eriksson UJ, Borg LA. Diabetes and embryonic malformations. Role of substrate-induced free-oxygen radical production for dysmorphogenesis in cultured rat embryos. Diabetes 1993;42:411–419.[Abstract]

30. Inoguchi T, Sonta T, Tsubouchi H et al. Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: Role of vascular NAD(P)H oxidase. J Am Soc Nephrol 2003;14:S227–S232.[Abstract/Free Full Text]

31. Han C, Demetris AJ, Liu Y et al. Transforming growth factor-β (TGF-β) activates cytosolic phospholipase A₂ (cPLA₂ )-mediated prostaglandin E₂ (PGE)₂/EP1 and peroxisome proliferator-activated receptor-gamma (PPAR-)/Smad signaling pathways in human liver cancer cells. A novel mechanism for subversion of TGF-β-induced mitoinhibition. J Biol Chem 2004;279:44344–44354.[Abstract/Free Full Text]

32. Martínez J, Moreno JJ. Role of Ca2⁺-independent phospholipase A₂ on arachidonic acid release induced by reactive oxygen species. Arch Biochem Biophys 2001;392:257–262.[CrossRef][Medline]

33. Jawerbaum A, Gonzalez ET, Sinner D et al. Diminished PGE₂ content, enhanced PGE₂ release and defects in ³H-PGE₂ transport in embryos from overtly diabetic rats. Reprod Fertil Dev 2000;12:141–147.[CrossRef][Medline]

34. Higa R, Gonzalez E, Pustovrh MC et al. PPAR and its activator PGI2 are reduced in diabetic embryopathy: Involvement of PPAR activation in lipid metabolic and signalling pathways in rat embryo early organogenesis. Mol Hum Reprod 2007;13:103–110.[Abstract/Free Full Text]

35. Meade EA, McIntyre TM, Zimmerman GA et al. Peroxisome proliferators enhance cyclooxygenase-2 expression in epithelial cells. J Biol Chem 1999;274:8328–8334.[Abstract/Free Full Text]

36. Jawerbaum A, Capobianco E, Pustovrh C et al. Influence of peroxisome proliferator-activated receptor activation by its endogenous ligand 15-deoxy 12,14 prostaglandin J2 on nitric oxide production in term placental tissues from diabetic women. Mol Hum Reprod 2004;10:671–676.[Abstract/Free Full Text]

37. Capobianco E, Jawerbaum A, Romanini MC et al. 15-Deoxy-12,14-prostaglandin J2 and peroxisome proliferator-activated receptor (PPAR ) levels in term placental tissues from control and diabetic rats: Modulatory effects of a PPAR agonist on nitridergic and lipid placental metabolism. Reprod Fertil Dev 2005;17:423–433.[CrossRef][Medline]

38. Zhang J, Fu M, Zhu X et al. Peroxisome proliferator-activated receptor is up-regulated during vascular lesion formation and promotes post-confluent cell proliferation in vascular smooth muscle cells. J Biol Chem 2002;277:11505–11512.[Abstract/Free Full Text]

39. Xu L, Han C, Wu T. A novel positive feedback loop between peroxisome proliferator-activated receptor-delta and prostaglandin E₂ signaling pathways for human cholangiocarcinoma cell growth. J Biol Chem 2006;281:33982–33996.[Abstract/Free Full Text]

40. Burdick AD, Bility MT, Girroir EE et al. Ligand activation of peroxisome proliferator-activated receptor-β/ (PPAR β/) inhibits cell growth of human N/TERT-1 keratinocytes. Cell Signal 2007;19:1163–1171.[CrossRef][Medline]

41. Han C, Wu T. Cyclooxygenase-2-derived prostaglandin E₂ promotes human cholangiocarcinoma cell growth and invasion through EP1 receptor-mediated activation of the epidermal growth factor receptor and Akt. J Biol Chem 2005;280:24053–24063.[Abstract/Free Full Text]

42. Tang CH, Yang RS, Fu WM. Prostaglandin E₂ stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase C, and c-Src pathway in primary cultured rat osteoblasts. J Biol Chem 2005;280:22907–22916.[Abstract/Free Full Text]

43. Ishibashi R, Tanaka I, Kotani M et al. Roles of prostaglandin E receptors in mesangial cells under high-glucose conditions. Kidney Int 1999;56:589–600.[CrossRef][Medline]

44. Alvarez-Soria MA, Largo R, Sanchez-Pernaute O et al. Prostaglandin E₂ receptors EP1 and EP4 are up-regulated in rabbit chondrocytes by IL-1β, but not by TNF. Rheumatol Int 2007;27:911–917.[CrossRef][Medline]

45. Regan JW. EP2 and EP4 prostanoid receptor signaling. Life Sci 2003;74:143–153.[CrossRef][Medline]

46. Honda A, Sugimoto Y, Namba T et al. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem 1993;268:7759–7762.[Abstract/Free Full Text]

47. Han HJ, Heo JS, Lee YJ. Estradiol-17β stimulates proliferation of mouse embryonic stem cells: Involvement of MAPKs and CDKs as well as protooncogenes. Am J Physiol Cell Physiol 2006;290:C1067–C1075.[Abstract/Free Full Text]

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