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Am J Physiol Cell Physiol 290: C1067-C1075, 2006. First published November 16, 2005; doi:10.1152/ajpcell.00222.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Ho Jae Han, Jung Sun Heo, and Yun Jung Lee

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

Submitted 9 May 2005 ; accepted in final form 8 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although the importance of estradiol-17beta (E2) in many physiological processes has been reported, to date no researchers have investigated the effects of E2 on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E2 on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E2 (10–9 M) significantly increased [3H]thymidine incorporation at >4 h and that E2 (>10–12 M) induced an increase of [3H]thymidine incorporation after 8-h incubation. Moreover, E2 (>10–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E2 stimulated estrogen receptor (ER)-{alpha} and -beta protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E2-induced increases in [3H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E2-BSA; 10–9 M) increased [3H]thymidine incorporation at >1 h, and E2-BSA (>10–12 M) increased [3H]thymidine incorporation after 1-h incubation. E2-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E2-BSA-induced increase of [3H]thymidine incorporation. Also, E2 and E2-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E2 increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E2 decreased the levels of p21cip1 and p27kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10–5 M PD-98059 (MEK inhibitor). Moreover, E2-induced increase of [3H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17beta stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes.

cyclin-dependent kinase; mitogen-activated protein kinase

EMBRYONIC STEM (ES) cells are derived from the inner cell mass of blastocysts, which have the potential to remain undifferentiated or to proliferate indefinitely in vitro under specific culture conditions (2). Recently, Brännvall et al. (4) showed that estrogen, via the estrogen receptor (ER), affects the proliferation and differentiation of neural stem cells, probably in conjunction with other factors governing the development of neural stem cells but not ES cells. Sung et al. (47) also demonstrated the presence of some reproductive hormones, such as luteinizing hormone, FSH, estrogen, progesterone, and testosterone, in undifferentiated murine ES cells and embryonic bodies. The biological effects of estrogen are mediated by two ER subtypes, ER-{alpha} and ER-beta. Recent data have provided increasing support for the notion that positive or negative regulation of cell growth by estrogen is, in part, a function of the ER subtype expressed in target cells (21, 22). In addition, the functionality of the reported embryonic ER has not been evaluated extensively, specifically as it relates to potential regulatory roles in embryonic growth or development or both. Furthermore, no researchers to date have investigated the effects of estradiol-17beta (E2) on the proliferation of ES cells.

In normal tissues, the frequency with which cells enter the cell cycle is tightly regulated by check or restriction points, which cannot be passed unless a stringent set of conditions is met, including the presence of mitogenic signals such as estrogen. The key factors that regulate progression through the cell cycle are a family of proteins called cyclins and cyclin-dependent kinases (CDKs) (51). Alternatively, evidence of cross-talk between steroid hormone receptors and signal transduction pathways has increased recently. Previous researchers observed that protein kinase activators enhance the transcriptional activity of ERs, suggesting that changes in cellular phosphorylation state should be important in determining the biological effectiveness of estrogen-occupied ERs (23, 53). Several groups have reported that E2 induces a rapid activation of MAPK/ERK in breast cancer cells, putting into doubt the initial consensus that the mitogenic effects of estrogens rely on transcriptional mechanisms (14, 44). Moreover, the effect of E2 on proliferation of murine ES cells and its related signaling molecules have yet to be determined.

In the present study, we used mouse ES (ES-E14TG2a) cells that were cultured in DMEM supplemented with a leukemia-inhibitory factor (LIF) to maintain an undifferentiated state and to support the derivation and expansion of ES cells (15, 46). These ES cells provide an attractive in vitro model in which to study their initial developmental and molecular mechanisms during embryonic growth and development. Thus this study was performed to investigate the effect of E2 on proliferation and its related signaling pathways in mouse ES cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Mouse ES cells were obtained from the American Type Culture Collection (ES-E14TG2a and ES-J1 cell lines; Manassas, VA). Experiments were conducted with ES-E14TG2a cells except in the studies indicated. FBS was obtained from BioWhittaker (Walkersville, MD). E2, estradiol-6-O-carboxymethyl oxime-BSA (E2-BSA; 32-to-1 estradiol-to-BSA ratio), tamoxifen, and PD-98059 were obtained from Sigma Chemical (St. Louis, MO). [3H]Thymidine was obtained from NEN (Boston, MA). Rabbit anti-ER-{alpha}, ER-beta, cyclin D1, cyclin E, CDK2, CDK4, p21cip1, and p27kip1 PAbs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho-p44/42 and p44/42 MAPK MAbs 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).

E2-BSA conjugate preparation. To eliminate potential artifacts due to contamination of commercially available E2-BSA (Sigma Chemical) preparations with unconjugated E2 (usually between 3 and 5%), 400 µl of E2-BSA were added to a centrifugation filter unit with a molecular mass cutoff of Mr 3,000 (Millipore, Billerica, MA) and centrifuged at 14,000 g until 50 µl of retained E2-BSA remained. The retained E2-BSA was washed three times with 350 µl of buffer and recovered, and then the volume was adjusted to 400 µl. In the present study, a 10–3 M stock solution of E2-BSA conjugate (Sigma Chemical) was prepared in DMEM and stored at –20°C. On the day of the experiment, the stock solution was supplemented in DMEM to a final concentration of 1 x 10–13 to 1 x 10–6 M E2-BSA (equivalent to 32 x 10–13 to 32 x 10–6 M free E2 concentration because 1 molecule of BSA is conjugated with 32 molecules of E2). Because the final concentration of BSA in the medium supplemented with E2-BSA was between 1 x 10–13 and 1 x 10–6 M, the control medium was supplemented with BSA to obtain an equivalent concentration. Values obtained by performing RIA of the three fractions (load, filtrate, and retenate) demonstrated that free, immunoassayable E2 (iE2) was present in the load (20 ± 3 ng/mg BSA conjugate for E2-BSA) and filtrate (2 ± 1 ng/mg BSA conjugate for E2-BSA), whereas iE2 was not detected in the retenate. The lower concentration of iE2 in the filtrate relative to the load resulted from loss of residual iE2 during the three washing steps. No E2-BSA was observed in the filtrate on the basis of protein assays or silver staining of SDS-PAGE (data not shown). This analysis suggests that fresh and untreated solutions of E2-BSA contain considerable amounts of free iE2 that can be removed by simple centrifugation through an Mr 3,000-cutoff filter cartridge.

ES cell culture. Murine ES cells were cultured with or without a feeder layer in the DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 3.7 g/l sodium bicarbonate, 1% penicillin and streptomycin, 1.7 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 5 ng/ml mouse LIF, and 15% FBS. For each experiment, cells were grown on gelatinized 12-well plates or 60-mm-diameter culture dishes in an incubator maintained at 37°C with 5% CO2. The medium was removed and replaced with serum-free DMEM, including all supplements containing LIF, for 12 h before the experiments. After that step, the cells were washed twice with PBS and then maintained in serum-free DMEM, including all supplements and indicated agents.

Alkaline phosphatase staining. Cells were washed twice with PBS and fixed for 15 min with 4% formaldehyde (in PBS) at room temperature. Cells were washed with PBS and incubated with a Sigma alkaline phosphatase substrate solution [200 µg/ml naphthol AS-MX phosphate, 2% N,N-dimethylformamide, 0.1 M Tris (pH 8.2), and 1 mg/ml Fast Red TR salt (4-chloro-2-methylbenzenediazonium salt; ZnCl)] for 10 min at room temperature. After being washed with PBS, the cells were photographed.

Immunofluorescent staining with SSEA-1. Cells were fixed and treated with MAb against mouse stage-specific embryonic antigen-1 (SSEA-1, 1:50 dilution; Santa Cruz Biotechnology) and then incubated for 30 min with FITC-conjugated secondary antibody raised in rabbit against mouse IgM (1:100 dilution). Fluorescence images were obtained using a fluorescence microscope (FluoView 300; Olympus, Tokyo, Japan).

[3H]Thymidine incorporation. [3H]Thymidine incorporation experiments were conducted as described by Brett et al. (5). Briefly, immediately before the study, the medium was changed to serum-free DMEM. One microcurie of methyl-[3H]thymidine (specific activity, 74 GBq/mmol or 2.0 Ci/mmol; Amersham Biosciences, Little Chalfont, UK) was added to each of the cultures. The incubation with [3H]thymidine continued for 1 h at 37°C. The 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 using a liquid scintillation counter (LS 6500; Beckman Instruments, Fullerton, CA). Values were converted from absolute counts to percentages of control values to allow comparison between experimental groups.

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

BrdU incorporation. Incorporation of the [3H]thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) was measured to determine DNA synthesis. ES cells were starved of serum before E2 stimulation. Subsequently, ES cells were treated with E2 for 8 h, 15 µM BrdU was 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 MAb (1:200 dilution; Molecular Probes, OR) in 2% BSA-PBS was incubated overnight at 4°C. After being washed with PBS, coverslips were mounted with Dako fluorescent mounting medium and plated onto glass slides using 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.

RNA isolation and RT-PCR. Total RNA was extracted from cells using STAT-60, monophasic solution of phenol and guanidine isothiocyanate obtained from Tel-Test (Friendswood, TX). Reverse transcription was conducted with 3 µg of RNA using a reverse transcription kit (AccuPower RT PreMix; Bioneer, Daejeon, Korea) with oligo(dT)18 primers. Next, 5 µl of RT products were amplified using a PCR kit (AccuPower PCR 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'-CGTTGCAGACTGAGATTGCC-3' (sense) and 5'-ACCGGACAGGTCCACATCTG-3' (antisense) for c-fos (356 bp), 5'-AACTCGGACCTTCTCACGTCG-3' (sense) and 5'-TGCTGAGGTTGGCGTAGACC-3' (antisense) for c-jun (355 bp), 5'-TCCATTCCGAGGCCACAGCAAG-3' (sense) and 5'-TCAGCTCGTTCCTCCTCTGACG-3' (antisense) for c-myc (266 bp), and 5'-CGTGAGACTTTGCAGCCTGA-3' (sense) and 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct-4 (519 bp). beta-actin PCR was also performed as a control to determine RNA quantity. RT-PCR products were separated and visualized using 1.2% agarose gels. To analyze the PCR products, we calculated the quantitative values of the E2 treatment group compared with the control group and then standardized these data relative to beta-actin.

Western blot analysis. Cells were harvested, washed twice with PBS, and lysed with buffer [20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml aprotinin, 1 mM PMSF, and 0.5 mM sodium orthovanadate] for 30 min on ice. The lysates were then cleared by performing centrifugation (10 min at 15,000 rpm and 4°C). Protein concentration was determined using the Bradford procedure (3). Equal amounts of protein (20 µg) were resolved by performing electrophoresis on 10% SDS-PAGE gels and transferred onto nitrocellulose membrane. After the blots were washed with TBST [10 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20], membranes were blocked with 5% skim milk for 1 h and incubated with the appropriate primary antibody at the dilutions recommended by the supplier. Subsequently, the membrane was washed, primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase, and the bands were visualized using ECL (Amersham Pharmacia Biotech, Little Chalfont, UK).

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


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of E2 on proliferation. Among the characteristics of pluripotent stem cells, we examined gene expression such as that of the carbohydrate epitope SSEA-1 (27), the POU domain transcription factor Oct-3/4 (41), and alkaline phosphatase activity (31). To confirm the undifferentiated state of mouse ES cells used in the present studies, alkaline phosphatase staining was conducted first. Mouse ES cells in the absence or presence of E2 for 24 h maintained alkaline phosphatase enzyme activity (Fig. 1A) and expressed SSEA-1 (a cell surface marker protein), which was detected using immunofluorescent staining (Fig. 1B). Oct-4 transcription factor is expressed in undifferentiated cells and downregulated upon differentiation (38). In this study, mouse ES cells treated with E2 for 24 h expressed levels of Oct-4 mRNA (Fig. 1C) and protein (Fig. 1D) equivalent to that of control cells. Therefore, the present results show that mouse ES cells maintained an undifferentiated state in the experimental conditions we used.


Figure 1
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  		Fig. 1. Characterization of mouse embryonic stem (ES) cells. A: alkaline phosphatase enzyme activity was measured in murine ES cells treated in absence or presence of estradiol-17beta (E2; 10–9 M) for 24 h as described in MATERIALS AND METHODS. B: immunofluorescent staining of murine ES cells treated in presence or absence of E2 with mouse stage-specific embryonic antigen-1 (SSEA-1) antibody. Scale bars, 20 µm. Magnification, x400. C: effect of E2 on Oct-4 (519 bp) and beta-actin (350 bp) mRNA expression levels. D: effect of E2 on Oct-4 and beta-actin protein expression levels. Bands represent 50–60 kDa of Oct-4 and 41 kDa of beta-actin.

 
DNA synthesis of mouse ES cells was determined on the basis of [3H]thymidine incorporation. Mouse ES cells were incubated with 10–9 M E2 for varying times (0–12 h) or for 8 h with various concentrations of E2 (0–10–6 M). As shown in Fig. 2A, a significant increase in [3H]thymidine incorporation was initially observed at 4 h after incubation with 10–9 M E2 (31% increase vs. control; P < 0.05). Figure 2B shows that 10–12 to 10–6 M E2 significantly increased [3H]thymidine incorporation after 8-h incubation. BrdU incorporation was also stimulated in response to E2 in a dose-dependent manner (Fig. 2C). Finally, there was a significant increase in cell number associated with various E2 concentrations (10–13 to 10–6 M) after 24-h incubation (Fig. 2D). To examine whether this effect of E2 is present in other mouse ES cells, the ES-J1 cell line was used. [3H]Thymidine incorporation was conducted for 8 h with various concentrations (0–10–6 M) of E2. As shown in Fig. 2E, a significant increase in [3H]thymidine incorporation was observed at 8 h after incubation with 10–9 M E2 (77% increase vs. control; P < 0.05). The largest increase in cell number was observed at a concentration of 10–9 M E2 (Fig. 2F). In addition, using Western blot analysis, we examined whether ER-{alpha} and ER-beta were present in mouse ES cells. ER-{alpha} and ER-beta were expressed in mouse ES cells, and their levels were significantly increased by E2 (Fig. 3A). To further investigate transcriptional regulation of E2, RT-PCR of c-fos, c-jun, and c-myc was conducted. As shown in Fig. 3B, E2 significantly stimulated protooncogene transcription of mouse ES cells, and these increases were inhibited by tamoxifen (antiestrogen; 10–6 M), suggesting that E2 significantly stimulates proliferation of mouse ES cells via ERs. To further investigate whether nuclear ERs are involved in E2-induced increase of [3H]thymidine incorporation, tamoxifen was added for 30 min before treatment with E2 for 4 h. As shown in Fig. 3C, tamoxifen blocked E2-induced increase of [3H]thymidine incorporation.


Figure 2
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  		Fig. 2. Effects of E2 on [3H]thymidine incorporation. A: murine ES cells were incubated in the presence of E2 (10–9 M) for varying times (0–12 h) and subsequently pulsed with 1 µCi of [3H]thymidine for 1 h before being counted. B: murine ES cells were incubated for 8 h with various concentrations of E2 (0–10–6 M) and pulsed with 1 µCi of [3H]thymidine for 1 h. C: 5'-bromo-2'-deoxyuridine (BrdU)-positive cells in response to different concentrations of E2 (0–10–6 M) for 8 h. D: murine ES cells were treated with E2 (0–10–6 M) for 24 h, and the cells were then counted using a hemocytometer. E: murine ES (ES-J1) cell line was incubated for 8 h with various concentrations of E2 (0–10–6 M) and pulsed with 1 µCi of [3H]thymidine for 1 h. F: murine ES-J1 cells were incubated with E2 (0–10–6 M) for 24 h, and the cells were then counted using a hemocytometer. Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 

Figure 3
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  		Fig. 3. Expression of estrogen receptors (ERs)-{alpha} and -beta in murine ES cells. Mouse ES cells were incubated with E2 (10–9 M) for 24 h. A: total protein was extracted and blotted using antibodies against ER-{alpha} or ER-beta, respectively. Bottom: means ± SE of 3 experiments for each condition determined using densitometry relative to beta-actin. Proteins were expressed at 50–70 kDa. B: murine ES cells treated with E2 (10–9 M) for 1 h after 30-min preincubation with tamoxifen (10–6 M), and then c-fos, c-jun, and c-myc gene expression was analyzed using RT-PCR. Genes were expressed in 356 bp for c-fos, 355 bp for c-jun, 266 bp for c-myc, and 350 bp for beta-actin. Each of the examples shown is representative of 3 independent experiments. *P < 0.05 vs. control. C: murine ES cells were treated with E2 (10–9 M) for 4 h after 30-min preincubation with tamoxifen (10–6 M) and 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. E2.

 
To examine whether membrane associated receptors of E2 are involved in proliferation of mouse ES cells, cells were treated with E2-BSA (membrane-impermeable BSA-conjugated E2) at 10–9 M for varying times (0–12 h) or with various concentrations (0–10–6 M) for 1 h. A significant increase in [3H]thymidine incorporation with 10–9 M E2-BSA was observed at 1 h (72% increase vs. control; P < 0.05) (Fig. 4A), and 10–12 to 10–6 M E2-BSA significantly increased [3H]thymidine incorporation after incubation for 1 h (Fig. 4B). E2-BSA-induced increase of BrdU incorporation was consistent with [3H]thymidine incorporation (Fig. 4C). To confirm the action of E2-BSA on membrane ERs, tamoxifen was added for 30 min before being treated with E2-BSA. As shown in Fig. 4D, tamoxifen did not inhibit E2-BSA-induced increase of [3H]thymidine incorporation.


Figure 4
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  		Fig. 4. Effects of estradiol-6-O-carboxymethyl oxime-BSA (E2-BSA) on [3H]thymidine incorporation. A: murine ES cells were incubated in the presence of E2-BSA (10–9 M; 3.2 x 10–8 M free E2) for varying times (0–12 h) and subsequently pulsed with 1 µCi of [3H]thymidine for 1 h. B: murine ES cells were incubated for 1 h with various concentrations of E2-BSA (0–10–6 M) and pulsed with 1 µCi of [3H]thymidine for 1 h. C: BrdU-positive cells in response to different concentrations of E2-BSA (0–10–6 M) for 8 h. D: murine ES cells were treated with E2-BSA (10–9 M) for 1 h after 30-min preincubation with tamoxifen (10–6 M) and pulsed with 1 µCi of [3H]thymidine for 1 h. Control cells were incubated with BSA (10–9 M). Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 
Involvement of E2 on p44/42 MAPKs phosphorylation and CDK expression. In experiments conducted to examine the phosphorylation of p44/42 MAPKs, p44/42 MAPK phosphorylation in response to E2 was observed for 0–60 min. The maximum phosphorylation of p44/42 MAPKs appeared at 10 and 5 min after E2 and E2-BSA treatments, respectively (Fig. 5, A and B). To support the effect of E2 on proliferation of ES cells, we observed the effect of E2 on protein expression levels of cyclin D1, cyclin E, CDK4, and CDK2, which are considered to be critical factors in G1-S progression. E2 increased cyclin D1, cyclin E, CDK4, and CDK2 at 4 h. These increases were inhibited by PD-98059 (10–5 M) (Fig. 6, A and B). However, PD-98059 (10–5 M) alone did not affect the protein levels of cell cycle regulators. CDK inhibitors p21cip1 and p27kip1 were downregulated in response to E2, and PD-98059 blocked downregulation by both of these CDK inhibitors (Fig. 6C). E2-induced increase of [3H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor; 5x10–6 M) (Fig. 6D). These findings suggest that p44/42 MAPKs and CDKs are involved in the E2-induced increase in proliferation.


Figure 5
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  		Fig. 5. Effect of E2 on p44/42 MAPK phosphorylation. A: murine ES cells were incubated with E2 (10–9 M) for 0–60 min and with 20% serum (positive control) and then harvested. Total protein was extracted and blotted using antibody against phospho-p44/42 MAPKs or total p44/42 MAPKs. B: murine ES cells were stimulated with E2-BSA (10–9 M) for 0–30 min, and then total extracted proteins were detected using phospho-p44/42 MAPK or total p44/42 MAPK antibodies. Each of the examples shown is representative of 3 independent experiments. Data are means ± SE of 3 experiments for each condition and were determined on the basis of densitometry relative to total p44/42 MAPKs. *P < 0.05 vs. control.

 

Figure 6
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  		Fig. 6. Effect of E2 on cyclin D1, cyclin E, cyclin-dependent kinase (CDK)4, and CDK2 expression. Mouse ES cells were preincubated with PD-98059 (10–5 M) for 30 min before being subjected to E2 (10–9 M) treatment for 4 h. Total protein was extracted and blotted using antibodies against cyclin D1 and cyclin E (A), CDK4 and CDK2 (B), or p21cip1 and p27kip1 (C). Proteins were expressed at 35 or 55 kDa. Each of the examples shown is representative of 3 independent experiments. D: murine ES cells were treated with E2 (10–9 M) for 1 h after 30-min preincubation with PD-98059 or butyrolactone I (5 x 10–6 M) and pulsed with 1 µCi of [3H]thymidine for 1 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. E2.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study demonstrate that E2 stimulated proliferation of mouse ES cells and that this action is mediated by MAPKs, CDKs, or protooncogenes. Our findings also strongly suggest that E2 plays a pivotal role in stimulating the DNA synthesis of ES cells. The present effects of E2 measured at 10–10 to 10–7 M concentrations represent physiological actions, although the normal serum concentration of E2 depends to some extent on species. In premenopausal women, the serum concentration of E2 is ≤0.28 x 10–9 M in the follicular phase and ≤1.1 x 10–9 M in the luteal phase and can increase to 150 x 10–9 M in the third trimester of pregnancy (7). It is known that the primary action of estrogen is mediated by ERs, a member of a large superfamily of structurally and functionally related nuclear transcription factors (24, 25, 31). Conventional ERs are transcription factors that regulate gene expression, and these mechanisms may be mediated by a complex system of interacting signal molecules in each different cell type (37, 45). However, little is known about the involvement of ERs in proliferation of mouse ES cells, although many transcriptional factors, including nuclear receptors, regulate the functions of mouse ES cells. In the present study, we have observed that both ER-{alpha} and ER-beta were expressed in mouse ES cells, and the increased [3H]thymidine incorporation by E2 was blocked using tamoxifen. These findings mean that positive feedback between E2 and ER-{alpha} and -beta amplified initial DNA synthesis by E2 in mouse ES cells. This control system must be able to make appropriate adjustments in the activity of the mouse ES cells to the desired value. The present results are in accord with a study in which the researchers reported that E2-induced neural ES cell proliferation was inhibited in the presence of ICI 182,780 (an ER antagonist) (4). The present results are also consistent with the observation that the stroma of the ER-{alpha}-knockout uterus did not proliferate in response to estrogen, indicating that the ER-dependent pathway is necessary to stimulate proliferation (10).

Among the first candidate genes to be investigated as potential targets of estrogen-induced mitogenesis was the immediate/early gene c-myc (13). It has also been shown that both ERs can interact with fos/jun to form a complex that augments transcription at activator protein-1 sites (1). Our results also show that E2 rapidly induced protooncogenes (c-myc, c-fos, and c-jun) in mouse ES cells. Lobenhofer et al. (29) reported that ICI 182,780 completely inhibited this induction but that none of the signal transduction inhibitors, such as PKC, phosphatidylinositol 3-kinase, and p44/42 MAPK inhibitors, had any effect on estrogen-induced myc and fos expression, suggesting that evocation of these genes is directly dependent on the activation of ERs. In contrast, Maggiolini et al. (30) demonstrated that immediate/early c-fos gene induction was inhibited not only by ICI 182,780 and 4-hydroxytamoxifen but also by PD-98059 in MCF-7 cells, suggesting that E2-induced proliferative events may be accomplished independently of ERs. Differently from genomic effects, the receptors mediating the rapid response of E2 have not yet been identified at the molecular level. To produce rapid nongenomic effects, ERs are thought to be located in or near the plasma membrane, where they can access the mechanisms of signal generation. At the same time, the activity constants measured for nongenomic effects of E2 varying from the subnanomolar to high micromolar concentration range clearly point to the diversity of these membrane sites as well as the complexity of their functioning (42). Several studies have suggested that membrane ERs can associate with membrane rafts and caveolae (6, 40). Consequently, we hypothesized that both genomic and nongenomic signaling molecules may be involved in E2-induced increase in the proliferation of mouse ES cells.

Indeed, various reports have suggested that E2 interacts with cell surface binding sites and induces intracellular signals, such as activation of adenylate cyclase, PLC, PKC, or ERK (26, 50). Multiple signaling pathways are rapidly stimulated in target cells that express endogenous ER-{alpha} and -beta, and these pathways have been linked to discrete cellular actions of E2 (54). The present results demonstrate that both plasma membrane-associated ERs and activation of MAPKs are involved in the E2-induced increase of proliferation. In ER-{alpha}- and ER-beta-deficient CHO-K1, COS-7, and Rat2 fibroblast cell lines, E2-induced activation of PKC or ERK was not blocked by ICI 182,780 or by antibodies against ER-{alpha} and -beta, which led the investigators to propose that a novel plasma membrane-associated ER mediates E2-induced activation of these cascades (36).

The downstream targets of estrogen-mediated MAPK activation are just beginning to be explored. The central role of E2-induced cell cycle control mechanisms are cyclin D1-CDK4 and cyclin E-CDK2, which phosphorylate substrates, including the product of the retinoblastoma susceptibility gene pRB, thereby allowing the initiation of DNA synthesis (17). In the present study, we have demonstrated that E2-induced cyclin D1-CDK4 and cyclin E-CDK2 stimulation was abrogated by PD-98059. Consistent with our present results, in another study (30), PD-98059 prevented E2-induced cyclin D1 accumulation in human thyroid tumor cells, which is an important intermediary in inducing cell cycle cascade. In contrast, p21 and p27 are known members of the Cip/Kip family of cell cycle regulators that inhibit the activation of cyclins A-, E-, and D-dependent CDKs (43). The present results also show that E2 downregulated both p21 and p27 and that PD-98059 blocked these inhibitions. Indeed, estrogen treatment relieved the inhibitory activity of p21 toward cyclin E-CDK2 as a result of a decrease in newly synthesized p21, which has greater inhibitory activity than preexisting p21 (11, 48). A potent inhibitor of CDK2, p27, is also involved in the assembly of cyclin D-CDK4 complexes. Alternatively, E2 may alter the properties of the inhibitors, reducing their ability to bind to the complex, or perhaps as a result of increased c-myc expression, inducing proteins that inhibit the interactions of p21 and p27 with cyclin E-CDK2 complexes (39). All of these results suggest that the E2-induced increase in the proliferation of mouse ES cells may be regulated by the integration of nuclear and cell surface signaling. Further studies are needed to identify other E2-related signal molecules in mouse ES cells. In conclusion, estradiol-17beta stimulates the proliferation of mouse ES cells, which is mediated by p44/42 MAPKs, CDKs, or protooncogenes.


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 ABSTRACT
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This research was supported by Grant SC 2210 from the Stem Cell Research Center of the 21st Century Frontier Research Program, which is funded by the Ministry of Science and Technology, Republic of Korea.


   FOOTNOTES
 

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)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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