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

摘要

During human trophoblast differentiation, inhibin alpha subunit mRNA expression and protein secretion are increased. To understand how inhibin alpha subunit gene was regulated during syncytialization, we firstly cloned the inhibin alpha 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 alpha promoter was the transcription factor activating protein 2 (AP2). Western blotting using specific antibodies against AP2alpha and AP2gamma confirmed that AP2alpha was the main subtype present in trophoblast cells, while AP2gamma 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 AP2alpha mRNA and protein during in vitro trophoblast differentiation correlated with an increase in inhibin alpha subunit gene expression. Furthermore, AP2alpha and AP2gamma overexpression in these cells was responsible for an increase in inhibin alpha subunit mRNA expression. We conclude that AP2 regulates the inhibin alpha subunit gene expression during trophoblast differentiation and may be a key regulator of syncytialization.