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GROWTH, DIFFERENTIATION, AND APOPTOSIS

EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca²⁺ influx and p44/42 MAPKs

Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Submitted 25 March 2005 ; accepted in final form 15 August 2005

	ABSTRACT

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We examined the effect of EGF on the proliferation of mouse embryonic stem (ES) cells and their related signal pathways. EGF increased [³H]thymidine and 5-bromo-2'-deoxyuridine incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). Inhibition of EGFR tyrosine kinase with AG-1478 or herbimycin A, inhibition of PLC with neomycin or U-73122, inhibition of PKC with bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²⁺ channels with nifedipine or methoxyverapamil prevented EGF-induced [³H]thymidine incorporation. PKC-, -_I, -, -, and - were translocated to the membrane and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased in response to EGF. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²⁺]_i. EGF also increased inositol phosphate levels, which were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and pretreatment with antioxidant (N-acetyl-L-cysteine) inhibited EGF-induced increase of [Ca²⁺]_i. In addition, we observed that p44/42 MAPK phosphorylation by EGF and inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²⁺ channels blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs with PD-98059 (MEK inhibitor) attenuated EGF-induced increase of [³H]thymidine incorporation. Finally, inhibition of EGFR tyrosine kinase, PKC, Ca²⁺ channels, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4, respectively. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation.

calcium; epidermal growth factor; mitogen-activated protein kinases; protein kinase C

However, there is little information concerning the regulatory mechanisms of cell proliferation by EGF in ES cells. The present study used mouse ES cells as a model for cell proliferation by EGF. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain conditions (10). ES cells were cultured in DMEM supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state and to support the derivation and expansion of ES cells (14, 47). These cells closely resemble their in vivo counterparts and thus provide a stable in vitro model of embryo growth and development and a tool with which a specific signaling system can be investigated. Thus this study was performed to investigate the effect of EGF on cell proliferation and its related signaling pathways in mouse ES cells.

	MATERIALS AND METHODS

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Materials. Mouse ES cells were obtained from the American Type Culture Collection (ES-E14TG2a). Fetal bovine serum was obtained from BioWhittaker (Walkersville, MD). EGF, EGTA, BAPTA-AM, AG-1478, herbimycin A, genistein, neomycin, U-73122, nifedipine, methoxyverapamil, PD-98059, SB-203580, N-acetyl-L-cysteine (NAC), A-23187, FITC-conjugated goat-anti mouse IgM, and -actin were obtained from Sigma (St. Louis, MO). [³H]thymidine and [³H]inositol phosphates were obtained from NEN (Boston, MA). Fluo-3 AM was obtained from Molecular Probes (Eugene, OR). Anti-pan-PKC was obtained from Upstate Biotechnology (Charlottesville, VA). Anti-PKC-, -_I, -, -, and -, cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). EGF receptor (EGFR), phospho-EGFR, phospho-p44/42 MAPKs, p44/42 MAPKs, phospho-p38 MAPK, and p38 MAPK antibodies were obtained from New England Biolabs (Hitchin, UK). Goat anti-rabbit IgG was obtained from Jackson Immunoresearch (West Grove, PA). All other reagents were obtained commercially and were of the highest purity available. Liquiscint was obtained from National Diagnostics (Parsippany, NJ).

ES cell culture. Mouse ES cells were cultured in DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 3.7 g/l of sodium bicarbonate, 1% penicillin and streptomycin, 1.7 mM L-glutamine, 0.1 mM -mercaptoethanol, 5 ng/ml mouse LIF, and 15% FBS with or without a feeder layer and cultured for 5 days in standard medium plus LIF. Cells were grown on gelatinized 12-well plates or 60-mm culture dishes in an incubator maintained at 37°C with 5% CO₂. The medium was changed to serum-free DMEM with LIF before the experiments.

Alkaline phosphatase staining. Mouse ES cells were washed twice with PBS and fixed for 15 min with 4% formaldehyde (in PBS) at room temperature. After being washed with PBS, cells were incubated with an alkaline phosphatase substrate solution (200 µg/ml naphthol AS-MX phosphate, 2% N,N-dimethylformamide, 0.1 M Tris, and 1 mg/ml Fast Red TR salt) for 10–15 min at room temperature and were washed with PBS. A photograph of these cells was taken.

Immunofluorescence staining with stage-specific embryonic antigen-1. Cells were fixed and treated with monoclonal antibody against mouse stage-specific embryonic antigen-1 (SSEA-1, 1:50; Santa Cruz Biotechnology) and then incubated for 30 min with FITC-conjugated second antibodies raised in rabbit against mouse IgM (1:100). Fluorescence images were obtained with a fluorescence microscope (fluoview 300, Olympus).

[³H]thymidine incorporation. [³H]thymidine incorporation was examined as described by Brett et al. (6). Briefly, ES cells were starved of serum before stimulation with EGF, and immediately before the study began the medium was changed to DMEM to further supplement EGF without serum. ES cells were incubated either in a medium containing EGF or in a medium that did not contain EGF for 24 h and were pulsed with 1 µCi of [methyl-³H]thymidine for 1 h at 37°C. The ES cells were then washed twice with PBS, fixed in 10% TCA at 23°C for 15 min, and then washed twice with 5% TCA. The acid-insoluble material was dissolved in 2 N NaOH for 12 h at 23°C. Aliquots were removed to determine radioactivity with a liquid scintillation counter (LS 6500, Beckman Instruments, Fullerton, CA). All values are means ± SE of triplicate experiments. Values were converted from absolute counts to a percentage of the control (EGF) to allow for comparison between experiments.

Bromodeoxyuridine incorporation. Incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was conducted to determine DNA synthesis. ES cells were grown in DMEM medium and were starved of serum before stimulation with EGF. The ES cells were treated with EGF for 24 h, 15 µM BrdU was then added, and the incubation continued for an additional 1 h. After several washes with PBS, cells were fixed with methanol [10% (vol/vol) for 10 min at 4°C] followed by incubation in 1 N HCl for 30 min at room temperature. The cells were then washed and incubated with 0.1 M sodium tetraborate for 15 min. Alexa Fluor 488-conjugated mouse anti-BrdU Ab (diluted 1:200; Molecular Probes) in 2% BSA-PBS was incubated overnight at 4°C. After a PBS wash, coverslips were mounted with Dako fluorescent mounting medium onto glass slides with Gelvatol and examined under a microscope (fluoview 300, Olympus). The number of BrdU-labeled cells relative to the total number of cells per field of vision was determined. A minimum of 10 fields of vision per coverslip were counted.

Measurement of intracellular Ca²⁺ concentration. Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored by using fluo-3 AM, which was initially dissolved in dimethyl sulfoxide and stored at –20°C. Mouse ES cells in 35-mm culture dishes were rinsed twice with a bath solution [in mM: 140 NaCl, 5 KCl, 1 CaCl₂, 0.5 MgCl₂, 10 glucose, 5.5 HEPES (pH 7.4)], incubated in the bath solution containing 3 µM fluo-3 AM with 5% CO₂-95% O₂ at 37°C for 40 min, rinsed two more times with the bath solution, mounted on a perfusion chamber, and scanned every second with a confocal microscope (x400; fluoview 300, Olympus). Fluorescence was excited at 488 nm, and emitted light was observed at 515 nm. All analyses of [Ca²⁺]_i were processed in a single cell, and results were expressed as relative fluorescence intensity.

Inositol phosphate formation assay. The assay performed in this study was a modified version of that described by Berridge et al. (3). Cells were labeled with myo-[³H]-inositol (2.5 µCi/ml, 2 ml final) for 24 h, followed by the addition of 10 mM LiCl for 15 min, and were treated with the appropriate agent. The medium was removed, cells were scraped off the dish in 1.2 ml H₂O and extracted in 1.8 ml of chloroform-methanol (1:2, vol/vol), and the upper phase was applied to AG 1-X8 columns (Bio-Rad Laboratories, Hercules, CA). After the cells were washed several times with 5 mM inositol and H₂O, the fraction containing the [³H]inositol phosphates [inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate (InsP₃)] was eluted with 1 M ammonium formate and 0.1 N formic acid.

H₂O₂ release. H₂O₂ levels were determined by a modified version of the method described by Zhou et al. (49). The cells were washed twice with ice-cold PBS and harvested by microcentrifugation and then resuspended in a Krebs-Ringer phosphate solution [KRPG (in mM): 145 NaCl, 5.7 sodium phosphate, 4.86 KCl, 0.54 CaCl₂, 1.22 MgSO₄, 5.5 glucose (pH 7.35)]. One hundred microliters of the reaction mixture [50 µM Amplex Red reagent containing 0.1 U/ml horseradish peroxidase (HRP) in KRPG] was added into each microplate well and then incubated at 37°C for 10 min. The reaction was initiated by adding the resuspended cells in 20 µl of KRPG. Fluorescence readings became stable within 30 min of the start of the reaction. The fluorescence intensities of reaction mixtures were measured at 30 min with a fluorescence microplate reader (Multiskan, Thermo Labsystems, Franklin, MA) equipped for absorbance at 560 nm.

Preparation of cytosolic and total membrane fractions. Preparation of cytosolic and total membrane fractions was performed by a modified version of the method described by Mackman et al. (27). The DMEM of mouse ES cells was exchanged at 48 h before experiments. The medium was then removed, and the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A [in mM: 137 NaCl, 8.1 Na₂HPO₄, 2.7 KCl, 1.5 KH₂PO₄, 2.5 EDTA, 1 dithiothreitol, and 0.1 PMSF, with 10 µg/ml leupeptin (pH 7.5)]. The resuspended cells were then mechanically lysed on ice by trituration with a 21.1-gauge needle. The lysates were first centrifuged at 1,000 g for 10 min at 4°C. The supernatants were centrifuged at 100,000 g for 1 h at 4°C to prepare cytosolic and total particulate fractions. The supernatants (cytosolic fraction) were then precipitated with 5 vols of acetone, incubated for 5 min on ice, and centrifuged at 20,000 g for 20 min at 4°C. The resultant 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 by the Bradford procedure (5).

RNA isolation and RT-PCR. Total RNA was extracted from mouse ES cells with STAT-60, a monophasic solution of phenol and guanidine isothiocyanate from Tel-Test (Friendwood, TX). Reverse transcription was conducted with 3 µg RNA and a reverse transcription system kit (AccuPower RT PreMix) with oligo(dT)18 primers. Five microliters of RT products was then amplified with a PCR kit (AccuPowerPCR PreMix), followed by denaturation at 94°C for 5 min and 30 cycles at 94°C for 45 s, 55°C for 1 min, and 72°C for 1 min followed by a 5-min extension at 72°C. The primers were 5'-CGTGAGACTTTGCAGCCTGA-3' (sense) and 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct-4 (519 bp). PCR of -actin was also performed as a control for quantity of RNA.

Western blot analysis. Cell homogenates (20 µg protein) were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After the blots were washed with 10 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20, the membranes were blocked with 5% skimmed milk for 1 h and incubated with the appropriate primary antibody at dilutions recommended by the supplier. The membrane was then washed, primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to HRP, and the bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Chalfont St. Giles, UK).

Statistical analysis. Results are expressed as means ± SE. All experiments were analyzed by ANOVA followed by, in some experiments, a comparison of treatment means with the control by the Bonferroni-Dunn test. Differences were considered statistically significant when P < 0.05.

	RESULTS

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Effect of EGF on cell proliferation. To confirm the undifferentiated state of mouse ES cells used in the experiments, we examined undifferentiated markers of stem cells, including alkaline phosphatase activity, carbohydrate epitope SSEA-1 expression, and Oct-4 (POU domain transcription factor). As shown in Fig. 1, A and B, mouse ES cells in both the presence and the absence of EGF maintained alkaline phosphatase enzyme activity and expressed SSEA-1, which was detected by immunofluorescent staining. Oct-4 transcription factor is expressed in undifferentiated cells and downregulated on differentiation (36). In this study, mouse ES cells treated with EGF expressed levels of Oct-4 mRNA (Fig. 1C) and protein (Fig. 1D) equivalent to those in the control cells. Therefore, the present results show that mouse ES cells maintained an undifferentiated state under our experimental conditions.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Characterization of mouse embryonic stem (ES) cells. A: alkaline phosphatase enzyme activity was measured in mouse ES cells treated in the presence or absence of EGF (100 ng/ml) as described in MATERIALS AND METHODS. B: immunofluorescent staining of mouse ES cells treated in the presence or absence of EGF with stage-specific embryonic antigen-1 (SSEA-1)-specific antibody. Scale bars, 20 µm (magnification, x400). C: Oct-4 (519 bp) and -actin (350 bp) mRNA expression levels of mouse ES cells in the presence or absence of EGF. Mouse embryonic fibroblasts (MEF) were used as a negative control. D: Oct-4 and -actin protein expression levels of mouse ES cells in the presence or absence of EGF. MEF were used as a negative control. Bands represent 50–60 kDa of Oct-4 and 41 kDa of -actin.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effects of EGF on [3H]thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and the phosphorylation of EGF receptor (EGFR). A: mouse ES cells were incubated in the presence of EGF (100 ng/ml) for various periods of time (0–24 h) under serum-free conditions and subsequently pulsed with 1 µCi of [3H]thymidine for 1 h before counting. B: mouse ES cells were incubated with various concentrations of EGF (0–200 ng/ml) for 24 h and pulsed with 1 µCi of [3H]thymidine for 1 h. C: BrdU-positive nuclei in response to different concentrations of EGF (0–200 ng/ml) for 24 h. D: total lysates of mouse ES cells treated with EGF (100 ng/ml) for 5 min were subjected to SDS-PAGE and blotted with phospho-EGFR or total EGFR antibody. The example shown is representative of 3 experiments. Bottom: means ± SE of 3 experiments for each condition determined from densitometry relative to total EGFR. E: mouse ES cells were pretreated with AG-1478 (10–5 M) or herbimycin A (10–6 M) for 30 min before EGF treatment for 24 h and then pulsed with 1 µCi of [3H]thymidine for 1 h. Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

Involvement of PLC/PKC in EGF-induced cell proliferation. Involvement of PLC in EGF-induced increase of cell proliferation was examined. Neomycin (10^–4 M) or U-73122 (10^–6 M) (PLC inhibitors) blocked EGF-induced increase of [³H]thymidine incorporation (Fig. 3A). EGF also increased inositol phosphate formation by 79 ± 8% compared with control at 90 s and gradually decreased (Fig. 3B). In addition, AG-1478 or herbimycin A blocked EGF-induced increase of inositol phosphates (Fig. 3C). We also investigated whether PKC is involved in EGF-induced cell proliferation. Bisindolylmaleimide I or staurosporine (PKC blockers, 10^–7 M) blocked EGF-induced increase of [³H]thymidine incorporation (Fig. 4A). The translocation of PKC from the cytosolic compartment to the membrane compartment was observed at 1 h after treatment with 100 ng/ml EGF (Fig. 4B). EGF enhanced the translocation of PKC-, -_I, -, -, and - isoforms to the membrane (Fig. 4C). Moreover, EGF-induced PKC activation was blocked by AG-1478 or herbimycin A (Fig. 4D).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effect of PLC blockers on EGF-induced increase of [3H]thymidine incorporation and effect of EGF on inositol phosphate (IP) formation. A: mouse ES cells were pretreated with neomycin (10–4 M) or U-73122 (10–6 M) for 30 min before EGF (100 ng/ml) treatment for 24 h and then pulsed with 1 µCi of [3H]thymidine for 1 h. B: inositol phosphate formation was measured after treatment with EGF for various periods of time (0–120 s). C: mouse ES cells were pretreated with AG-1478 (10–5 M) or herbimycin A (10–6 M) for 30 min before EGF treatment for 90 s, and then inositol phosphate formation was measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of EGF on PKC activation. A: mouse ES cells were pretreated with bisindolylmaleimide I or staurosporine (10–7 M) for 30 min before EGF (100 ng/ml) treatment for 24 h and then pulsed with 1 µCi of [3H]thymidine for 1 h. Values are means ± SE of 4 independent experiments with triplicate dishes. B and C: pan-PKC protein and PKC-, -I, -, -, and - isoforms that were present in either the cytosolic compartment or the membrane compartment were detected by Western blotting as described in MATERIALS AND METHODS. Bands represent 80 kDa of pan-PKC (B), 80–90 kDa of PKC-, -I, -, -, and - (C), and 41 kDa of -actin. Each example shown is a representative of 3 experiments. D: mouse ES cells were pretreated with AG-1478 (10–5 M) or herbimycin A (10–6 M) for 30 min before EGF treatment for 1 h, and then pan-PKC protein was detected in membrane compartment. B and D, bottom: means ± SE of 3 experiments for each condition determined from densitometry relative to -actin. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Effect of EGF on the alteration of intracellular Ca2+ concentration ([Ca2+]i). A: mouse ES cells were loaded with 2 µM fluo 3-AM in serum-free medium for 40 min and treated with EGF (100 ng/ml). B–E: mouse ES cells were pretreated with EGTA (4x10–3 M, B), BAPTA-AM (10–5 M, C), nifedipine (10–6 M, D), or methoxyverapamil (10–6 M, E) for 30 min, and then EGF-induced Ca2+ influx was measured. F: mouse ES cells were pretreated with nifedipine or methoxyverapamil (10–6 M) for 30 min before EGF treatment for 24 h and then pulsed with 1 µCi of [3H]thymidine for 1 h. G–N: mouse ES cells were pretreated with herbimycin A (10–6 M, G), genistein (10–6 M, H), neomycin (10–4 M, I), U-73122 (10–6 M, J), bisindolylmaleimide I (10–7 M, K), staurosporine (10–7 M, L), or N-acetyl-L-cysteine (NAC, 10–5 M, N) for 30 min before EGF (100 ng/ml) treatment. Changes of [Ca2+]i were monitored by confocal microscope and expressed as relative fluorescence intensity (RFI). A-23187 (10–6 M) was used as positive control. Level of intracellular H2O2 was also determined as described in MATERIALS AND METHODS. M: mouse ES cells were incubated with EGF for different time periods (0–60 min). The examples shown are a representative of 5 experiments. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Effect of EGF on the activation of MAPKs. Mouse ES cells were treated with EGF (100 ng/ml) for various periods of time (0–90 min). A and B: phosphorylation of p44/42 MAPKs (A) and p38 MAPK (B) were detected as described in MATERIALS AND METHODS. The example shown is a representative of 3 experiments. C: mouse ES cells were pretreated with PD-98059 or SB-203580 (10–6 M) for 30 min before EGF treatment for 24 h and then pulsed with 1 µCi of [3H]thymidine for 1 h. Values are means ± SE of 4 independent experiments with triplicate dishes. D: mouse ES cells were pretreated with AG-1478 (10–5 M), herbimycin A (10–6 M), neomycin (10–4 M), bisindolylmaleimide I (10–7 M), nifedipine (10–6 M), or PD-98059 (10–6 M) for 30 min before treatment with EGF for 30 min. Phosphorylation of p44/42 MAPKs was then detected. The example shown is representative of 3 experiments. A, B, and D, top: means ± SE of 3 experiments for each condition determined from densitometry relative to total p44/42 MAPKs. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

View larger version (44K): [in this window] [in a new window]   Fig. 7. Effect of AG-1478, bisindolylmaleimide I, nifedipine, and PD-98059 on EGF-stimulated level of cell cycle regulators. Mouse ES cells were pretreated with AG-1478 (10–5 M), bisindolylmaleimide I (10–7 M), nifedipine (10–6 M), or PD-98059 (10–6 M) for 30 min before incubation of EGF (100 ng/ml) for 4 h, and then total lysates were subjected to SDS-PAGE and blotted with cyclin D1 (A), cyclin E (B), cyclin-dependent kinase (CDK)2 (C), or CDK 4 (D) antibody. The example shown is a representative of 4 experiments. A–D, top: means ± SE of 4 experiments for each condition determined from densitometry relative to -actin. *P < 0.05 vs. control; **P < 0.05 vs. EGF alone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

The activation of EGFR tyrosine kinase by binding of EGF leads to intracellular signaling, usually cell proliferation. In the present study, we demonstrated that the selective EGFR tyrosine kinase blockers AG-1478 and herbimycin A completely inhibited the EGF-induced stimulation of cell proliferation. This suggests that EGF stimulates tyrosine kinase activity, resulting in a rapid phosphorylation of its own receptor (22). Therefore, the onset of the signal seems to require an increase of tyrosine kinase activity linked to EGFR. Previous reports showed that EGFR signaling is necessary for normal craniofacial development (31) and ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during embryonic pancreatic development (21) in an Egfr-deficient (Egfr–/–) mouse model. A PLC binding site in the kinase domain of EGFR has been identified, giving the receptor the capacity to signal through PLC- and Ca²⁺ (33). The link between EGF and PLC- activation in ES cells, however, is not clear. Cross et al. (11) reported an activation of PLC, as measured by [³H]inositol phosphate production, in porcine endothelial cells overexpressing EGF. However, this pathway does not appear to be linked to MAPK activation and cell proliferation. In addition to PLC- phosphorylation, increased PKC activity has been observed in response to EGF. Furthermore, previous studies demonstrated that EGF-dependent mitogenesis is associated with the activation of phospholipid-dependent PKC in normal mammary epithelial cells (4), which may be consistent with the results of this study of PKC translocation from the cytosolic compartment to the membrane compartment in mouse ES cells. In the present results, PKC-, -_I, -, -, and - isoforms translocated to the membrane compartment in response to EGF in mouse ES cells. In previous reports, PKC isoforms such as , , , , and were expressed in mouse ES cells (39, 44), and despite different cell types, EGF induced the distribution of PKC-, -_I, and - isoforms to cell membranes for cell protection in Caco-2 cells (2). Among the PKC family, conventional PKCs (PKC-, -_I, -_II, -) require increases of both [Ca²⁺]_i and diacylglycerol (DAG) (26, 32). Moreover, it was reported that Ca²⁺-dependent PKC- and - are involved in proliferation signals, whereas Ca²⁺-independent PKC- and - are involved in survival pathways of colorectal tumor cells (20). The present results may also suggest that Ca²⁺-dependent as well as Ca²⁺-independent PKCs are involved in EGF-induced mouse ES cell proliferation.

In addition, Ca²⁺ is one of the key regulators of DNA synthesis and cell proliferation, being responsible for calmodulin-mediated activation of transcription factors either in the cytoplasm or in the nucleus (8). Therefore, in EGF-stimulated cells, it is possible that gene transcription could continue as long as the Ca²⁺ signaling does not stop. This feature could explain the requirement for growth factors in the extracellular medium as long as the cells progress into a proliferative state. In the present study, we found that EGF induces increases in [Ca²⁺]_i levels in mouse ES cells and the influx of Ca²⁺ from the extracellular medium is required to sustain the spiking activity, as shown by the addition of EGTA. In the present study, EGF-induced [Ca²⁺]_i increase was also blocked by the preincubation of BAPTA-AM, which means that EGF stimulates the release of Ca²⁺ from intracellular Ca²⁺ pools, correlated with the increase of inositol phosphate levels. Furthermore, we found that the addition of nifedipine or methoxyverapamil partially inhibited this increase. Therefore, it appears that the EGF-induced Ca²⁺ influx is mediated by voltage-dependent Ca²⁺ channels in mouse ES cells. In A431 human epidermoid carcinoma cells, EGF increased [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, with a lower and shorter increase of intracellular Ca²⁺ in the absence of extracellular Ca²⁺ (13). EGF also increased [Ca²⁺]_i by both Ca²⁺ release and Ca²⁺ influx in NIH3T3 cells expressing EGFR (42), which is consistent with the results of this study in mouse ES cells. The present results also show that PKC blockers inhibited EGF-induced Ca²⁺ influx. Previous studies showed that the activation of PKC affects intracellular Ca²⁺ levels in various cell types (17, 41), which is also consistent with the results of this study.

Because it has been reported that EGF-induced intracellular H₂O₂ plays an essential role in the increase of intracellular Ca²⁺ in Rat-2 fibroblasts (23), we investigated whether H₂O₂ is also involved in the increase of [Ca²⁺]_i by EGF in mouse ES cells. Consistent with previous reports, EGF rapidly increased the production of H₂O₂. In addition, incubation with NAC completely inhibited EGF-induced increase of [Ca²⁺]_i. These results suggested that EGF-induced intracellular H₂O₂ formation is responsible for the increase of [Ca²⁺]_i, which is involved in EGF-induced stimulation of mouse ES cell proliferation.

Next, we examined regulation of the MAPK cascade by EGFR in ES cells and the roles of PLC- and Ca²⁺ in these pathways. Despite different cell systems, many previous reports have demonstrated that EGF stimulates cell proliferation correlated with the magnitude of p44/42 MAPK activation (9). Consistent with previous reports, we observed the phosphorylation of p44/42 MAPKs by EGF in a time-dependent manner and the attenuation of this activation by AG-1478, herbimycin A, neomycin, bisindolylmaleimide I, nifedipine, and PD-98059, which may indicate the signal pathway of EGF-induced p44/42 MAPK activation. Yoon et al. (48) reported that the activation of EGFR tyrosine kinase resulted in p44/42 MAPK activation in human cholangiocarcinoma cells. Regulation of the MAPK cascade and endothelial cell proliferation by PLC- has been suggested by previous studies. Xia et al. (46) reported that VEGF stimulation led to PLC- tyrosine phosphorylation, inositol phosphate production, and cell proliferation in porcine aortic endothelial cells. PKC is also known to activate MAPK through activation of the serine threonine kinase Raf-1 (29). Transforming growth factor-1-induced activation of the Raf-MEK-MAPK signaling pathway via a PKC-dependent mechanism was examined in embryonic rat lung fibroblasts (1). The downstream activation of MAPKs was found to be sensitive to inhibition by PD-98059, which is consistent with previous findings in primary endothelial cells (40). Furthermore, PD-98059 has been shown to inhibit DNA synthesis driven by VEGF and FGF in these cells (30), which is consistent with the results of this study. On the other hand, one of the downstream targets of EGF-mediated MAPK activation is cell cycle regulators, which phosphorylate substrates including the product of the retinoblastoma susceptibility gene pRB, thereby allowing initiation of DNA synthesis (15). Previous reports have shown that PD-98059 prevented EGF-induced stimulation of cyclin D1 in human thyroid tumor cells, which is an important intermediate in inducing the cell cycle cascade (28). In the present study, we demonstrated that EGF-induced stimulation of cyclin D1, cyclin E, CDK2, and CDK4 was blocked by AG-1478, bisindolylmaleimide I, nifedipine, and PD-98059. Therefore, we suggest that EGF activates receptor tyrosine kinase, which stimulates the PKC/Ca²⁺-MAPK pathway, and finally these cascades lead to mouse ES cell proliferation (Fig. 8). On the basis of all of the results of this study, we would suggest that the ES cell property of self-renewal depends on stoichiometric balance among various signal molecules, and an imbalance in any one of these molecules can cause ES cell identity to be lost. However, all of these are not exclusively expressed by pluripotent ES cells and can be found in other types of cells in the soma. Therefore, the potential role of ES cells in maintaining pluripotence or self-renewal remains to be determined. In conclusion, EGF partially stimulates proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPK signal pathways through EGFR tyrosine kinase phosphorylation.

View larger version (30K): [in this window] [in a new window]   Fig. 8. The hypothesized model for the signal pathways involved in EGF-induced ES cell proliferation. EGF activates receptor tyrosine kinase, which stimulates PLC to generate inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). In turn, DAG activates PKC, which stimulates MAPK activation, continuously inducing ES cell proliferation. Receptor tyrosine kinase or PKC also increases H2O2 formation, subsequently leading to the increase of [Ca2+]i. In another pathway, InsP3 stimulates the release of Ca2+ from an intracellular Ca2+ pool, and to sustain the spiking activity Ca2+ influx from an extracellular medium is required. Finally, Ca2+ stimulates ES cell proliferation. TK, tyrosine kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; InsP3R, InsP3 receptor; ER, endoplasmic reticulum. The solid line is the proposed pathway, and the dashed line is suspected pathway.

	GRANTS

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Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (e-mail: hjhan{at}chonnam.ac.kr)

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