Wilms' Tumor Suppressor Gene (WT1) Suppresses Apoptosis by Transcriptionally Downregulating BAX Expression in Immature Rat Granulosa Cells

Overview

Journal J Ovarian Res

Publisher Biomed Central

Specialties Gynecology & Obstetrics
Reproductive Medicine

Date 2014 Dec 11

PMID 25491731

Citations 3

Authors

Minji Park

Yuri Choi

Hyeonhae Choi

Jaesook Roh

Affiliations

Soon will be listed here.

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.

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).

Citing Articles

Whole exome sequencing identified a rare WT1 loss-of-function variant in a non-syndromic POI patient.

Wang Y, Chen Q, Zhang F, Yang X, Shang L, Ren S Mol Genet Genomic Med. 2021; 10(1):e1820.

PMID: 34845858 PMC: 8801142. DOI: 10.1002/mgg3.1820.

rno-miR-128-3p promotes apoptosis in rat granulosa cells (GCs) induced by norepinephrine through Wilms tumor 1 (WT1).

Li M, Xue L, Xu W, Liu P, Li F In Vitro Cell Dev Biol Anim. 2021; 57(8):775-785.

PMID: 34554376 PMC: 8585816. DOI: 10.1007/s11626-021-00609-y.

Wilms' tumor in a 51-year-old patient: An extremely rare case and review of the literature.

Hu J, Jin L, He T, Li Y, Zhao Y, Ding Y Mol Clin Oncol. 2016; 4(6):1013-1016.

PMID: 27313862 PMC: 4888037. DOI: 10.3892/mco.2016.839.

References

Antonsson B, Martinou J . The Bcl-2 protein family. Exp Cell Res. 2000; 256(1):50-7. DOI: 10.1006/excr.2000.4839. View

Ito K, Oji Y, Tatsumi N, Shimizu S, Kanai Y, Nakazawa T . Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway. Oncogene. 2006; 25(30):4217-29. DOI: 10.1038/sj.onc.1209455. View

Markstrom E, Svensson E, Shao R, Svanberg B, Billig H . Survival factors regulating ovarian apoptosis -- dependence on follicle differentiation. Reproduction. 2002; 123(1):23-30. DOI: 10.1530/rep.0.1230023. View

Harman S, LOUVET J, ROSS G . Interaction of estrogen and gonadotrophins on follicular atresia. Endocrinology. 1975; 96(5):1145-52. DOI: 10.1210/endo-96-5-1145. View

Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63. DOI: 10.1016/0022-1759(83)90303-4. View

Hirshfield A . Effect of a low dose of pregnant mare's serum gonadotropin on follicular recruitment and atresia in cycling rats. Biol Reprod. 1986; 35(1):113-8. DOI: 10.1095/biolreprod35.1.113. View

Rauscher 3rd F, Morris J, Tournay O, Cook D, Curran T . Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence. Science. 1990; 250(4985):1259-62. DOI: 10.1126/science.2244209. View

Hirshfield A . Development of follicles in the mammalian ovary. Int Rev Cytol. 1991; 124:43-101. DOI: 10.1016/s0074-7696(08)61524-7. View

Hughes Jr F, Gorospe W . Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology. 1991; 129(5):2415-22. DOI: 10.1210/endo-129-5-2415. View

10.

Tilly J, Kowalski K, Johnson A, Hsueh A . Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology. 1991; 129(5):2799-801. DOI: 10.1210/endo-129-5-2799. View

11.

Tilly J, Billig H, Kowalski K, Hsueh A . Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol. 1992; 6(11):1942-50. DOI: 10.1210/mend.6.11.1480180. View

12.

Oltvai Z, Milliman C, Korsmeyer S . Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993; 74(4):609-19. DOI: 10.1016/0092-8674(93)90509-o. View

13.

Kreidberg J, Sariola H, LORING J, Maeda M, Pelletier J, Housman D . WT-1 is required for early kidney development. Cell. 1993; 74(4):679-91. DOI: 10.1016/0092-8674(93)90515-r. View

14.

Williams G, Smith C . Molecular regulation of apoptosis: genetic controls on cell death. Cell. 1993; 74(5):777-9. DOI: 10.1016/0092-8674(93)90457-2. View

15.

Wang Z, Qiu Q, Enger K, Deuel T . A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1. Proc Natl Acad Sci U S A. 1993; 90(19):8896-900. PMC: 47467. DOI: 10.1073/pnas.90.19.8896. View

16.

Drummond I, Rupprecht H, Madden S, Rauscher 3rd F, Sukhatme V . DNA recognition by splicing variants of the Wilms' tumor suppressor, WT1. Mol Cell Biol. 1994; 14(6):3800-9. PMC: 358747. DOI: 10.1128/mcb.14.6.3800-3809.1994. View

17.

Tilly J, Tilly K, Kenton M, Johnson A . Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid.... Endocrinology. 1995; 136(1):232-41. DOI: 10.1210/endo.136.1.7828536. View

18.

Loeb D . WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members. Cell Cycle. 2006; 5(12):1249-53. DOI: 10.4161/cc.5.12.2807. View

19.

Roh J, Bae J, Lee K, Mayo K, Shea L, Woodruff T . Regulation of Wilms' tumor gene expression by nerve growth factor and follicle-stimulating hormone in the immature mouse ovary. Fertil Steril. 2008; 91(4 Suppl):1451-4. PMC: 2749456. DOI: 10.1016/j.fertnstert.2008.07.012. View

20.

Yoon O, Roh J . Regulation of FSH receptor expression by the Wilms' tumor 1 gene product (WT1) in immature rat granulosa cells. Mol Reprod Dev. 2012; 79(6):368. DOI: 10.1002/mrd.22036. View