Transcription factor AP2 regulates human inhibin subunit gene expression during in vitro trophoblast differentiation

Abstract

During human trophoblast differentiation, inhibin α subunit mRNA expression and protein secretion are increased. To understand how inhibin α subunit gene was regulated during syncytialization, we firstly cloned the inhibin α promoter and found a region with transcriptional activity related to the differentiation state. In this paper, we identified this protein and defined its DNA-binding site. Protein purification and identification were done by DNA affinity chromatography followed by mass spectrometry and western blotting. In order to confirm the identity of the protein, characterize its DNA-binding properties and to measure its expression during in vitro trophoblast differentiation, gel retardation assays and real-time polymerase chain reaction were done. We found that the cytotrophoblastic protein interacting with the inhibin α promoter was the transcription factor activating protein 2 (AP2). Western blotting using specific antibodies against AP2α and AP2γ confirmed that AP2α was the main subtype present in trophoblast cells, while AP2γ was barely detectable. Supershift experiments indicated that these two factors were able to bind to the sequence 5'-GCCtcaAGC-3'. We also observed an increase in AP2α mRNA and protein during in vitro trophoblast differentiation correlated with an increase in inhibin α subunit gene expression. Furthermore, AP2α and AP2γ overexpression in these cells was responsible for an increase in inhibin α subunit mRNA expression. We conclude that AP2 regulates the inhibin α subunit gene expression during trophoblast differentiation and may be a key regulator of syncytialization.