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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Stem Cells. Author manuscript; available in PMC Feb 10, 2010.
Published in final edited form as:
PMCID: PMC2818886
NIHMSID: NIHMS168007

Oct 4 is Critical for Survival/Antiapoptosis of Murine Embryonic Stem Cells Subjected to Stress. Effects Associated with STAT3/Survivin

Abstract

Understanding survival/anti-apoptosis of murine embryonic stem (ES) cells may enhance their clinical potential. We hypothesized that Oct-4 might be involved in survival of undifferentiated ES cells under stress. Oct-4 tetracycline conditional knockout cell line ZHBtc4 was used to test this possibility and apoptosis was induced by either etoposide, heat shock or UV exposure. Apoptosis in Oct-4 knocked-down ES cells was significantly increased in response to all stress situations compared to parental cells. Oct-4 knockdown was not associated with changes in morphology, or expression of nanog, SSEA-1, KLF-4, or Sox2 within the time-frame and culture conditions used, suggesting that enhanced sensitivity of these cells to apoptosis was not due to an overtly differentiated state of the cells. To address potential intracellular mediators, we focused on IAP family member Survivin, an anti-apoptosis protein. The Survivin promoter was transfected into ES cells after knock-down of Oct-4. Survivin promoter activity was dramatically decreased in the Oct-4 knock-down cells. Western blots substantiated that Oct-4 knockdown ES cells had decreased Survivin protein expression. Since the Survivin promoter does not have binding sites for Oct-4, this suggested an indirect effect of Oct-4 on expression of Survivin. LIF-induced STAT3 is responsible for ES cell survival, and STAT3 regulates Survivin expression in breast cancer cells. Western Blot analysis showed that down regulated Oct-4 was associated with decreased phosphorylation of STAT3. Our results suggest that Oct-4 is essential for anti-apoptosis of ES cells in response to stress, effects that may be mediated through the STAT3/Survivin pathway.

Keywords: Mouse Embryonic Stem Cells, Apoptosis, Etoposide, UV, Heat Stress, Oct 4, Stat3, Survivin

Introduction

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM)1. They are capable of undergoing unlimited numbers of symmetrical divisions without differentiation; in addition they can give rise to differentiated cell types that are derived from all three primary germ layers of the embryo1. They can colonize germ lines resulting in chimeric animals, undergo multilineage differentiation in vitro and produce a range of well-differentiated progenitors2, 3, suggesting a potential for ES cells in cell replacement and gene therapy. Apoptosis is critical for many biological events such as embryonic development, and knowledge of apoptosis of ES cells is important for future use of these cells. Oct-4, a POU homeobox transcription factor, is preferentially expressed and active in ES cells, and expression appears to be required for maintenance of the undifferentiated state of ES cells46. Our recent paper demonstrated that ES cells didn’t initiate apoptosis as it does in somatic cells, and this allowed an unusual tolerance to polyploidy7. This suggested to us that Oct-4 may be involved in the apoptotic process in ES cells.

Using a tetracycline-inducible murine ES cell line (ZHBtc4), and its parental control line (CGR8), we demonstrate that Oct4 is an important protector for survival of ES cells from apoptosis induced by Etoposide, UV, or heat shock. The Oct4 effects may be mediated through the Stat3/Survivin pathway as induced reduction of the expression of Oct4 was associated with decreased expression of Survivin and phosphorylated Stat3.

Materials and Methods

Cell culture and cell lines

Wild type ES cell line CGR8 and Oct-4 conditional Knockout cell line ZHBtc4 ( A gift from Dr Austin Smith, University of Edinburgh, Edinburgh, UK.) were cultured on gelatinized plates in Dulbecco’s Modified Eagle medium (DMEM) with 15% ES cell-qualified fetal calf serum (FCS; Hyclone, logan, UT), 5.5 × 10−2 mM β-mercaptoethanol, (Gibco BRL, Carlsbad, CA) and 103 U/ml LIF (Chemicon, Temecula, CA). Conditional knockout cell line ZHBtc4 is a modified CGR8 cell line, in which the native Oct-4 gene is replaced by a tetracycline regulated Oct-4 gene. Addition of tetracycline decreases expression of Oct-48 in the ZHBtc4, but not parental CGR8, ES cell line.

Apoptosis assay for ES cells after stress induction

Wild type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with/without tetracycline for 24 hours before stress induction. For etoposide stress: Etoposide (0, 0.5 and 2 ug/ml) was added and cells cultured for another 24 hours. For heat shock stress: ES cells were heated at 44°C for 20 min and cells were cultured an additional 24 hours at 37°C. For UV stress: ES cells were exposed to 20 J/m2, and cells were cultured an additional 24 hours at 37°C. Cells were collected and stained with Annexin V and activated Caspase 3 antibody (BD Bioscience, San Diego, CA.) for analysis of apoptosis.

Promoter activity analysis

Wild type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 cells were seeded in a 60 mm dishes (2–3 × 105 cells/dish) in 5 ml of complete DMEM growth medium (as described above) and grown to 50–80% confluence. Cells were cultured for 24 hours with/without tetracycline before transfection. ES cells were transfected with 10 ug /dish Survivin promoter vectors pluc or pluc13429 along with 1ug/dish of an internal control vector, using calcium phosphate transfection kit (BD Bioscience, San Diego, CA). Luciferase was analyzed 48 hours after transfection. Luciferase activity was normalized by Renila luciferase control vector.

Protein expression and Nanog RNA expression analysis after tetracycline treatment

Wild type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with/without tetracycline. Cells were collected at 24 and 48 hours after tetracycline treatment. In one additional group, cells were cultured with tetracycline for 24 hours and 0.5 ug/ml Etoposide was added to the media for another 24 hours. Cells were collected for extract RNA and total protein. Western blot analysis for anti Oct-4 (Santa cruz), phospho-STAT3 (Cell signaling), STAT3 (Cell signaling), and Survivin (R&D), was conducted. RT-PCR analysis for Nanog RNA expression was also performed. Primers for Nanog RNA were the RT2-PCR primer set from Superarray (Frederick, MD; proprietary sequence).

Flow analysis for KLF-4, SOX2 and SSEA-1 expression

Wild type ES cell line CGR8 and Oct-4 conditional knockout cell line ZHBtc4 were cultured with/without tetracycline. Cells were collected at 24 and 48 hours after tetracycline treatment. One portion of cells was collected and stained with SSEA-1 antibody (Santa Cruz, Santa Cruz, CA). The other portion of the cells was fixed by Cytoperm/CytoFix (BD Bioscience, San Diego, CA.) and stained with KFL-4- and Sox2-antibodies (Santa Cruz, Santa Cruz, CA). KLF, Sox2 and SSEA-1 expression are associated with the undifferentiated state of cells.10,11

Results and Discussion

Influence of Oct4 suppression on apoptosis of ES cell lines

To determine whether Oct-4 played a role in apoptosis of murine ES cell lines, we evaluated the effects of stress on parental ES cell line, CGR8, and on the parental cell line derived Oct-4 conditional knockout ES cell line, ZHBtc4, with and without first preincubating the CGR8 and ZHBtc4 cells with tetracycline. Addition of tetracycline to ZHBtc4, but not to CGR8, cells greatly reduced expression of Oct4 (Figure 1). Twenty-four hours after addition of tetracycline, ES cells were exposed to Etoposide-, UV-, or heat shock-induced stress. The ZHBtc4 cell line in which Oct4 expression was significantly decreased by pretreatment of these cells with tetracycline, demonstrated significantly increased apoptosis in response to Etoposide (Figure 2A, ,1B),1B), UV (Figure 2C) and heat shock (Figure 2C) compared to the CGR8 ES cells with or without tetracycline pretreatment, and the ZHBtc4 cells without tetracycline pretreatment. The ZHBtc4 ES cells subjected to tetracycline, and then to Etoposide still maintained an immature cell phenotype with no evidence of differentiation by morphology (Data not shown), and by expression of Nanog (Figure 3) which was similar to that of CGR8 cells with and without treatment with tetracycline and Etoposide, and to that of ZHBtc4 cells without tetracycline treatment, in the absence and presence of Etoposide. SSEA-1, KLF-4 and SOX2 were also checked after tetracycline treated for 24 and 48 hours. SSEA-1, KLF-4 and SOX2 are well known markers for undifferentiated ES cells.10,11 Our data showed that after treatment of ZHBtc4 cells with tetracycline for 24 and 48 hours, the expression levels of these markers were similar to that of the wild type cell CGR8. This strongly suggested that the treated ES cells were still in a relatively undifferentiated stage even after Oct-4 knock-down for 48 hours (Figure 4). The lack of noticeable differentiation of the cells during the time-frame studied and under the culture conditions used suggests that enhanced responsiveness of Oct-4 knock-down cells to induction of apoptosis is not due to the fact that effects were on differentiated cells. Thus, reduction of Oct4 expression in immature and undifferentiated ES cells (ZHBtc4) in the presence of tetracycline, enhanced apoptosis of ES cells in response to stresses induced by Etoposide, UV, and heat shock, suggesting that Oct4 plays a protective role in stress-induced apoptosis.

Figure 1
Oct-4 expression before and after tetracycline treatment
Figure 2Figure 2Figure 2
Characterization of stress-induced apoptosis in embryonic stem (ES) cells
Figure 3
Nanog mRNA expression
Figure 4Figure 4Figure 4
Expression of stem cell markers after Oct-4 deletion

Influence of Oct4 on expression of the IAP family member, Survivin

Survivin is an antiapoptotic protein12, 13, which is highly expressed in embryonic tissue1417. In order to see if Oct4 expression correlated with expression of Survivin, we evaluated activity of the Survivin promoter, in CGR8 and ZHBtc4 ES cells pretreated or not with tetracycline. Survivin luciferase promoter pluc 1342 and pluc were transfected into the two ES cell lines. The promoter pluc was used to assess the basal activity of the Survivin promoter. Tetracycline treatment did not have a significant effect on Survivin promoter activity of CGR8 cells, which was similar to the activity seen in both CGR8 and ZHBtc4 cells not exposed to tetracycline (Figure 5). However, the Survivin promoter activity was greatly reduced in the ZHBtc4 cells pre-treated with tetracycline (Figure 5). Therefore, decreased Survivin promoter activity directly correlated with tetracycline-induced decrease in expression of Oct4, suggesting that Oct4 directly or indirectly regulates Survivin expression. Since a binding region for Oct4 has not been reported in the Survivin promoter area18, it is likely that Oct4 may be indirectly involved in regulating expression of Survivin.

Figure 5
Survivin promoter activity

Another transcription factor, signal transducer and activator of transcription-3 (STAT3) has been implicated by others in regulation of apoptosis/survival in other cell types19, 20. These effects are mediated by Survivin2123. STAT3 is activated by phosphorylation of a single tyrosine residue (Tyr 705) by Janus Kinase and chimerizes via a reciprocal-SH2 phosphotyrosine interaction24. Chimeric Stat3 translocates to the nucleus and activates other genes24, 25. As seen in Figure 6, decreased phosphorylation of Stat3, but not effects on total Stat3 protein levels were manifest in ZHBtc4 cells in which tetracycline-induced suppression of Oct4 expression occurred. Phosphorylation of Stat3 was not influenced in CGR8 cells with or without tetracycline, or in ZHBtc4 cells without tetracycline. Thus, it is probable that decreased down-modulation of Survivin in ZHBtc4 cells induced by tetracycline, was a result of decreased phosphorylation of Stat3 associated with decreased Oct4 expression. A number of other Oct-4-regulated genes have been identified,26 and it is possible that changes in expression of some of these genes may also in some way be relevant to the enhanced sensitivity of the Oct-4 knock-down ES cells to induced differentiation of apoptosis.

Figure 6
Protein change after Oct-4 deletion

In summary, our data strongly suggest that Oct4 expression in mouse ES cells is required for protection from apoptosis induced by different apoptosis inducing stresses. Moreover, this protective effect is linked and may be mediated through the STAT3-Survivin pathway.

Acknowledgements

These studies were supported by U.S. Public Health Service Grants: RO1 HL67384, RO1 HL56416, and a Project in PO1 HL53586 (to H.E.B.) and RO1 CA102283 and HL75783 to R.A.H. The support of the Leukemia and Lymphoma Society (SCOR 7388-06) to R.A.H. is also acknowledged.

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