title: Wilms' tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells.
abstract: 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.
  
  
  CONCLUSIONS
  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).
authors.affiliation: Laboratory of Reproductive Endocrinology, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, San17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, South Korea. ovaries@hanmail.net.
authors.forename: Minji
authors.initials: M
authors.lastname: Park
authors.affiliation: Laboratory of Reproductive Endocrinology, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, San17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, South Korea. mintrabbit87@gmail.com.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Choi
authors.affiliation: Laboratory of Reproductive Endocrinology, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, San17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, South Korea. dalpu2002@naver.com.
authors.forename: Hyeonhae
authors.initials: H
authors.lastname: Choi
authors.affiliation: Laboratory of Reproductive Endocrinology, Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, San17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, South Korea. rohjaesook@hanyang.ac.kr.
authors.forename: Jaesook
authors.initials: J
authors.lastname: Roh
mesh_terms: D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D002470:Cell Survival; D002478:Cells, Cultured; D015536:Down-Regulation; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic; D025721:WT1 Proteins / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: C494828:Bax protein, rat; D025721:WT1 Proteins; D051028:bcl-2-Associated X Protein