Essential but differential role of FOXL2wt and FOXL2C134W in GDF-9 stimulation of follistatin transcription in co-operation with Smad3 in the human granulosa cell line COV434

• FOXL2 is required for GDF-9 activation of follistatin transcription in COV434 cells.
• FOXL2wt and FOXL2C134W similarly bind Smad3.
• In co-ordination with Smad3, FoxL2C134W prevents GDF-9 activity.
• FOXL2C134W altered activity requires intact Smad binding cis-element.

The FOXL2C134W mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2C134W negated GDF-9 stimulation of follistatin transcription. However, mutation of the Smad binding element (SBE) located in the intronic enhancer elements in the follistatin gene restored normal FOXL2 activity to FOXL2C134W, thus the altered activity of FOXL2C134W is dependent on the ability of Smad3 to directly bind the SBE. Mutation of the FOXL2 binding element (FBE) or the FBE and SBE completely prevented GDF-9 activity, suggesting that the FBE is essential for GDF-9 stimulation in COV434. Overall, our study supports the view that altered interaction of FOXL2C134W with co-factors may underlie the pathogenesis of this mutation in GCTs.