Oct-4 Is Critical for Survival/Antiapoptosis of Murine Embryonic Stem Cells Subjected to Stress: Effects Associated with Stat3/Survivin

Abstract

Understanding survival/antiapoptosis 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. The 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 with 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 the inhibitor of apoptosis proteins family member Survivin, an antiapoptosis protein. The Survivin promoter was transfected into ES cells after knockdown of Oct-4. Survivin promoter activity was dramatically decreased in the Oct-4 knockdown 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. Leukemia inhibitory factor-induced signal transducer and activator of transcription-3 (STAT3) is responsible for ES cell survival, and STAT3 regulates Survivin expression in breast cancer cells. Western blot analysis showed that downregulated Oct-4 was associated with decreased phosphorylation of STAT3. Our results suggest that Oct-4 is essential for antiapoptosis of ES cells in response to stress, effects that may be mediated through the STAT3/Survivin pathway.