Wilms’ tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells

Journal of Ovarian Research

Jaesook Roh¹  Hyeonhae Choi¹  Yuri Choi¹  Minji Park¹ 

[1] Laboratory of Reproductive Endocrinology, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, San17 Haengdang-dong, Seongdong-gu 133-791, Seoul, South Korea

关键词: Rat;    Granulosa cell;    Caspase;    bcl-2;    Bax;    Apoptosis;    WT1;   

Others  :  1136036
DOI  :  10.1186/s13048-014-0118-y

 received in 2014-07-03, accepted in 2014-12-02,  发布年份 2014

【 摘 要 】

Background

The important role of WT1 in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of inhibin-α and FSH receptor. There is also considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage.

Methods

GCs were obtained from small preantral follicles of immature rat ovary. Bax and bcl-2 mRNA and protein levels in GCs transfected with WT1 (−KTS) or WT1 (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with caspase 3/7 activity and TUNEL assay. The mechanism by which WT1 regulates Bax expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs.

Results

Here, we showed that WT1 (−KTS) suppressed endogenous Bax transcript and protein expression, and this inhibition resulted from direct binding of WT1 in the Bax promoter region in vivo. In addition, anti-apoptotic effects of WT1 (−KTS) were demonstrated based on MTT assays, a sensitive bioluminescence caspase 3/7 assay and TUNEL assays. On the other hand, WT1 has no role on bcl-2 expression in GCs.

Conclusion

These findings suggest that activation of WT1 is necessary for maintenance of GC survival during early stage of follicles and WT1 can play a role in protecting apoptosis through the regulation of upstream activator (Bax), as well as through regulation of downstream effecter (caspases 3 and 7).

【 授权许可】

   
2014 Park et al.; licensee BioMed Central.

【 图 表 】

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