Oct-4 Is Crucial for Stress-Induced Murine Embryonic Stem Cell Survival/Anti-Apoptosis Mediated by STAT3/Survivin.

Murine embryonic stem (ES) cells are pluripotent, and may be of potential use for cell replacement and gene therapy. By understanding the mechanism of survival and anti-apoptosis of ES cells, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that undifferentiated ES cells converted into polyploidy instead of apoptosis under the stress. However, differentiated ES cells went through apoptosis in the same manner as mature cells. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for choosing between polyploidy and apoptosis of ES cells under the stress. To address this question, Oct-4 tetracycline conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. To evaluate whether Oct-4 was essential for stress-induced apoptosis of murine ES cells, we used three methods to induce stress: etoposide treatment, heat shock and UV exposure. Four treatment groups were studied, A: Oct-4 knock down ZHBtc4; B: Control ZHBtc4; C: Tetracycline treated CGR8 cell line; D: Control CGR8. CGR8 is the wild type parental cell line for ZHBtc4. These latter two groups were added to eliminate potential effects of tetracycline. After knockdown of Oct-4 for 24 hours, we induced apoptosis by etoposide, heat shock or UV exposure. Apoptosis for Oct-4 knocked down ES cells was significantly increased in response to all stress situations. This suggests that Oct-4 is important for modulating stress-induced apoptosis of ES cells. In order to address potential mechanisms for these effects, we focused on one member of the IAP family—Survivin. Survivin is a well known anti-apoptosis protein. In addition, Oct-4 and survivin are both specifically expressed in embryonic tissue, germ line and tumor tissues (such as breast, pancreatic and colon cancer), but not in other tissues or at low levels in these cells. We hypothesized that the mechanism of Oct-4 related anti-apoptosis may be mediated through Survivin. Towards this possibility, we transfected the Survivin promoter into ES cells after knockdown of Oct-4. Promoter activity of the group with knocked down Oct-4 expression was dramatically decreased, indicating that Oct-4 mediated anti-apoptosis may act through Survivin. Western blots showed that Oct-4 knocked down ES cells had decreased Survivin protein expression. STAT3 is a well known transcription factor. Studies have shown that LIF-induced STAT3 is responsible for ES cell survival. Also, it was reported that STAT3 regulated survivin in breast cancer. We tested whether Oct-4 regulated Survivin through STAT3. Western Blot analysis showed that down regulated Oct-4 induced decreased phosphorylation of STAT3 and expression of total STAT3 protein. This suggests that Oct-4 is essential for anti-apoptosis of ES cells in response to stress, and this effect may be mediated through the STAT3/Survivin pathway.