GATA-4 is a granulosa cell factor employed in inhibin-α activation by the TGF-β pathway

Part of heterodimeric inhibin, inhibin-α is crucial for mammalian ovarian function. Regulation of inhibin-α expression in granulosa cells is both endocrine, primarily by follicle-stimulating hormone (FSH), and paracrine, primarily by members of the transforming growth factor β (TGF-β) superfamily. Smad proteins transmit TGF-β signals to the nucleus, but the cooperating transcription factors involved in inhibin-α promoter activation remain unknown. Transcription factor GATA-4 regulates inhibin-α in gonadal cells, and the FSH cascade activates GATA-4. We hypothesized that the TGF-β signalling cascade and GATA-4 also cooperate to regulate inhibin-α expression. In KK-1 granulosa tumour cells, which resemble normal granulosa cells and express inhibin-α, we found that TGF-β upregulated GATA-4 expression. Transient transfection experiments in KK-1 cells demonstrated that dominant negative GATA-4 variants or mutations of GATA-binding sites in the inhibin-α promoter attenuated TGF-β-induced gene activation. In GATA-4-deficient COS-7 cells, TGF-β enhanced the expression of the inhibin-α promoter only in the presence of exogenous GATA-4. Smad3, but not Smad2, cooperated with GATA-4 in the transcriptional activation of the inhibin-α promoter, and immunoprecipitation experiments in KK-1 cells revealed a physical Smad3:GATA-4 interaction. Our data suggest that GATA-4, interacting with Smad3, is a cofactor for TGF-β signalling to activate inhibin-α in granulosa cells.