Microarray analysis of Foxl2 mediated gene regulation in the mouse ovary derived KK1 granulosa cell line: Over-expression of Foxl2 leads to activation of the gonadotropin releasing hormone receptor gene promoter

Abstract

BackgroundThe Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary.MethodsTransiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software.ResultsMicroarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an activator of the GnRHR gene.ConclusionsPotential Foxl2 regulated ovarian genes have been identified through microarray analysis and comparison of these data to other microarray studies. The Foxl2 responsiveness of the GnRHR gene has been validated and provided evidence of Foxl2 transcriptional activation of the GnRHR gene promoter in the mouse ovary derived KK1 granulosa cell line.

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary

“…3E). Conversely, expression of the key negative transcriptional regulator of the majority of these enzymes, Foxl2 (Escudero et al, 2010), was increased (Fig. 3F).…”

“…Mouse models in which ERα is overexpressed generally develop hyperplastic glands and adenocarcinomas with a short latency (Frech et al, 2005;Tilli et al, 2003). We therefore hypothesized that estrogen production in MMTV-cre/Id4 f/f mice might be compromised in Id4-deficient mice to account for the ductal elongation defects.…”

FOXL2: a central transcription factor of the ovary

“…Its overexpression in GCT-derived cells upregulates pro-apoptotic genes, such as TNF-R1, FAS or TRAIL-R, receptors of proapoptotic ligands (Kim et al 2011). FOXL2 also activates the expression of the GNRH receptor in human and mouse GCs, which may play a pro-apoptotic role (Escudero et al 2010, Cheng et al 2013. The pro-apoptotic role of FOXL2 has mainly been studied in GCT cells, and has not been demonstrated in vivo yet.…”

“…The examination of FOXL2 transcriptional targets in various contexts suggests that FOXL2 may be important in many ovarian processes. Several transcriptomic studies on human GCT-derived cells or mice ovaries at different stages of development have described many potential targets of FOXL2 (Batista et al 2007, Garcia-Ortiz et al 2009, Uhlenhaut et al 2009, Escudero et al 2010, Rosario et al 2012. These studies are limited, in the case of GCT-derived cells by the fact that the mutation or extinction of FOXL2 can affect cell identity in the long-term, and 'in the case of mouse ovaries' by the major morphological changes associated with the absence of FOXL2 and by the superposition of direct and indirect effects on multiple cell types (Caburet et al 2012).…”

Notch gain of function in mouse periocular mesenchyme downregulates FoxL2 and impairs eyelid levator muscle formation, leading to congenital blepharophimosis

“…KK1 mouse granulosa tumour cell line was obtained from Kenneth W. Escudero (Escudero et al, 2010). 3T3 cells were maintained in DMEM supplemented with 10% FBS and 100 U/ml penicillin-streptomycin solution (Hyclone).…”