FOXL2C134W-Induced CYP19 Expression via Cooperation With SMAD3 in HGrC1 Cells

Germline knockout studies in female mice demonstrated an essential role for forkhead box L2 (FOXL2) in early follicle development, whereas an inducible granulosa cell (GC)-specific deletion of Foxl2 in adults has shown ovary-to-testis somatic sex reprogramming. In women, over 120 different germline mutations in the FOXL2 gene have been shown to cause blepharophimosis/ptosis/epicantus inversus syndrome associated with or without primary ovarian insufficiency. By contrast, a single somatic mutation (FOXL2(C134W)) accounts for almost all adult-type GC tumors (aGCTs). To test the hypothesis that FOXL2(C134W) differentially regulates the expression of aGCT markers, we investigated the effect of FOXL2(C134W) on inhibin B and P450 aromatase expression using a recently established human GC line (HGrC1), which we now show to bear two normal alleles of FOXL2. Neither FOXL2(wt) nor FOXL2(C134W) regulate INHBBmessenger RNA(mRNA) expression. However, FOXL2(C134W) selectively displays a 50-fold induction of CYP19 mRNA expression dependent upon activin A. Mechanistically, the CYP19 promoter is activated in a similar way by FOXL2(C134W) interaction with SMAD3, but not by FOXL2(wt). SMAD2 had no effect. Moreover, FOXL2(C134W) interactions with SMAD3 and with the FOX binding element located at -199 bp upstream of the ATG initiation codon of CYP19 are more sustainable than FOXL2(wt). Thus, FOXL2(C134W) potentiates CYP19 expression in HGrC1 cells via enhanced recruitment of SMAD3 to a proximal FOX binding element. These findings may explain the pathophysiology of estrogen excess in patients with aGCT.