Homeobox gene Distal-less 3 (DLX3) is a regulator of villous cytotrophoblast differentiation

Dlx3, a member of the large homeobox gene family of transcription factors, is important for murine placental development. Targeted deletion of Dlx3 in the mouse results in embryonic death due to placental failure. This study investigated the role of human DLX3 in villous cytotrophoblast (VCT) differentiation in the placenta. Primary VCT from human term placentae, which spontaneously differentiate when maintained in culture over 72 h, showed a significant increase in mRNA and protein expression of DLX3 and 3βHSD. The functional role of DLX3 was determined using trophoblast derived-cell line, BeWo. Forskolin treated BeWo cells showed significantly increased DLX3 mRNA and protein expression. Forskolin stimulation also showed a significant increase in syncytin and 3βHSD mRNA expression, and increased release of βhCG into the cell culture supernatant. To determine whether DLX3 had a direct or indirect effect on VCT differentiation, mRNA and protein expression of DLX3 was increased using a plasmid DLX3 over-expression construct. Over-expression of DLX3 resulted in increased mRNA expression of 3βHSD and syncytin, as well as increased secretion of β-hCG protein in the cell culture medium. In conclusion, we provide evidence that DLX3 acts upstream of syncytin, 3βHSD and βhCG and that DLX3 has a regulatory role in VCT differentiation.