Differential Apoptotic and Proliferative Activities of Wild-type FOXL2 and Blepharophimosis-ptosis-epicanthus Inversus Syndrome (BPES)-associated Mutant FOXL2 Proteins

Abstract

Abstract. FOXL2 is an essential transcription factor that is required for proper development of the ovary and eyelid. Mutations in FOXL2 cause an autosomal dominant genetic disorder, blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). BPES type I patients have eyelid malformation and premature ovarian failure leading to infertility, whereas women with type II BPES are fertile or subfertile. In the present study, we evaluated and compared apoptotic and antiproliferative activities of wild-type (WT) and mutant FOXL2 proteins found in BPES type I and II in human granulosa cell tumor-derived KGN cells. Ectopic expression of WT FOXL2 induced apoptosis and inhibited cell cycle progression in human granulosa cells. In contrast, mutated FOXL2s found in BPES type I significantly reduced these activities, whereas mutated FOXL2s in BPES type II showed intermediate activities. Furthermore, mutant FOX L2 proteins were defective in activating transcription of target genes including Caspase 8, FAS, p21, and BMP4, which regulate apoptosis, proliferation, and differentiation of granulosa cells. Thus, decreased apoptotic and antiproliferative activities caused by mutant forms of FOXL2 found in BPES patients may at least partially contribute to the pathophysiology of ovarian dysfunction. FOXL2 is a member of the forkhead (FH) family of transcription factors known to be required for proper development of the ovary and eyelid [1]. FOXL2 is a single-exon gene that encodes a protein of 376 amino acids containing a DNA-binding FH domain and an alanine-rich region. Mutations in the FOXL2 gene cause the autosomal dominant genetic disorder blepharophimosis-ptosis-epicanthus inversus syndrome (BPES; MIM 110100) [2]. BPES type I patients have eyelid malformation associated with premature ovarian failure (POF). In contrast, type II BPES patients show defects in eyelid development, but affected individuals are fertile [2]. Although FOXL2 mRNA is expressed in fetal, juvenile, and adult mouse ovaries, undifferentiated granulosa cells (GCs) of small follicles have prevalent FOXL2 expression [3]. Female Foxl2 knockout mice are infertile and have defects in granulosa cell differentiation [4,5]. Conditional knockout of Foxl2 in adult mice results in somatic transdifferentiation of an adult ovary into a testis [4]. Also, more than 97% of adult-type ovarian GC tumors have a somatic point mutation (C134W) in the FOXL2 gene [6]. These human and mouse studies indicate that FOXL2 is an essential protein required for proper development and maintenance of the ovary. Although the mechanism by which FOXL2 orchestrates ovarian physiology is not completely understood, its role in regulating target genes involved in apoptosis, the cell cycle, and differentiation has been reported [7][8][9][10].In the present study, we investigated the functional differences of FOXL2 mutants found in BPES type I and II patients and observed compromised activities of mutant FOXL2 proteins that may at least partly contribute to the pathophysiology of ovarian dysfunction...

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku

“…PCR products were digested with XhoI and EcoRI (Takara Bio, Shiga, Japan) and were ligated into p3XFLAG-CMV-10 (Sigma-Aldrich, St. Louis, MO, USA). Plasmids carrying Myc-tagged FOXL2 WT, C134W, ΔAla, ΔFH, FH, K36R, K48R, Q53X, I80T, I84S, N105S, and N109K were prepared 17,18,51 . Myc-tagged K124R, K150R, K246R, K124Q, F167X, and W204X expression plasmids were produced by recombinant PCR using the following primers: FOXL2-F (5′-CTAGAATTCAAATGATGGCCAGCTACCCC-3′),…”

“…Forkhead box protein L2 (FOXL2) is a member of the forkhead box (FOX) superfamily of transcriptional factors that possess a DNA-binding domain. Previous studies have found that FOXL2 regulates the transcription of critical genes involved in a wide spectrum of biological processes, including sex determination, steroidogenesis, differentiation, apoptosis, and proliferation [13][14][15][16][17][18][19] . FOXL2 is highly expressed in the ovaries and eyelids, and autosomal dominant germline mutations in FOXL2 cause blepharophimosis, ptosis epicanthus inversus syndrome (BPES) manifested by an eyelid malformation and primary ovarian insufficiency (POI) 20 .…”

Transcriptome sequencing revealed that knocking down FOXL2 affected cell proliferation, the cell cycle, and DNA replication in chicken pre-ovulatory follicle cells

“…In addition to the many studies on mammal granulosa cells have shown role of FOXL2 in apoptosis [2,34,35], we also observed that chicken FOXL2 knockdown decreased the cell apoptosis rate in the poGC. The role of FOXL2-regulation in the cell cycle in granulosa cells has also been published [7,31,34].…”

“…More than 95% of adult-type ovarian granulosa cell tumours (OGCTs) are highly associated with a somatic point mutation (C134W) in FOXL2, suggesting a potential relationship between FOXL2 and human granulosa cell function [6]. Further studies in humans and mice indicate that the normal FOXL2 protein induces GC apoptosis and inhibits cell proliferation, while the mutant protein compromises these activities, thus contributing to OGCTs [7,8].…”

Functional Analysis of a Novel FOXL2 Indel Mutation in Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I

“…To date, over 110 BPES-related mutations in over 210 families have been reported. Intragenic mutations of the FOXL2 gene make up the greatest proportion of the genetic defects detected in BPES, representing 71% of the group 7 . Frameshift mutations (most frequent), in-frame mutations, nonsense mutations and missense mutations are all observed in the FOXL2 gene.…”