FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells

Abstract

Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.

FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development

“…Our results indicate further that the GCTs in the Foxo1/3 and Foxo1/ 3Pten mutant mice exhibit features similar to hAGCTs and KGN cells (6,8,33) rather than to the juvenile GCTs and COV434 cells (6,40). Of particular relevance, the expression profiles for many of genes are similarly altered in the Foxo1/3/Pten GCTs, AGCTs, and KGN cells compared with the COV434 cells: AMH, FOXO1, and EMX2 are down-regulated; FOXL2, GATA4, SMAD3, INHBB, and SOX9 are present (17,18,33).…”

“…Of particular relevance, the expression profiles for many of genes are similarly altered in the Foxo1/3/Pten GCTs, AGCTs, and KGN cells compared with the COV434 cells: AMH, FOXO1, and EMX2 are down-regulated; FOXL2, GATA4, SMAD3, INHBB, and SOX9 are present (17,18,33). Furthermore, the intense immunostaining and colocalization of FOXL2 and pS-MAD2/3 in nuclei of cells within the AGCTs combined with the lack of PTEN and low/cytoplasmic localization of FOXO1 was similar to that in the GCTs of the Foxo1/3 and Foxo1/3Pten mutant mice.…”

“…GATA4 is not only required for gonadal ridge formation (11) but also for normal follicle development (30) and steroidogenesis (85,86). GATA4 is also present in hAGCTs (33,34,87) and appears to synergize with SMAD3 to promote tumor cell proliferation (33). Thus, GATA4 also appears to be a marker of AGCTs (88).…”

Acetylation of GATA4 on Lysine Residue K313 Promotes Osteoblastic Cells Growth

“…A previous study has pointed out that GATA4 could activate CCND2 promoter [32]. Herein, GATA4 acetylation was observed to increase CCND2 promoter activity, while GATA4 mutated at lys-313 showed low transactivation potential.…”

“…GATA4 is known to activate CCND2 promoter [32]. Herein, we overexpressed wt or mutated GATA4 and CBP or HDAC1 in 293T cells, and measured CCND2 promoter activity, to detect whether GATA4 acetylation will impact the targeting effects between GATA4 and CCND2.…”