Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts

Differentiation of primary villous cytotrophoblasts into syncytia is associ ated with increasing production of alpha and beta human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, w e present a detailed study on the mechanisms involved in the differentiatio n-dependent regulation of the trophoblast-specific CG alpha gene promoter. Site-directed mutations in each of the five DNA-elements of the composite e nhancer were performed to investigate the contribution of the individual re gulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show th at deletion of one cyclic AMP response element (CRE) did not affect CG alph a promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished t ranscription at early and later stages of in vitro differentiation. Upon mu tation the enhancer elements alpha ACT, JRE, and CCAAT significantly decrea sed luciferase reporter transcription; however their contribution to the to tal promoter activity did not change during in vitro differentiation. Contr ary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift, assay, the TSE interacted with activating protein (AP)-2 alpha in both primary trophoblas ts and choriocarcinoma cells. While CRE-interacting proteins were detectabl e 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response elemen t-binding protein (CREB)-1, and AP-2 alpha was observed on Western blots. M oreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show tha t CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2 alpha localized to the syncytium and some cytototrophoblasts as w ell as to stromal and endothelial cells of the placental villus. Phosphoryl ated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected i n syncytial nuclei, suggesting the presence of functional, cAMP-dependent t ranscriptional complexes in the differentiated tissue. In agreement to the in, vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CG alpha promo ter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAM P levels and overexpression of the catalytic subunit of protein kinase A. A dditionally, we demonstrate that overproduction of the enzyme enhanced prot ein expression and binding of AP-2 alpha to the TSE. We conclude that diffe rentiation-dependent transcription of the CG alpha gene in villous trophobl asts is mainly governed by increasing expression of AP-2a and PKA-dependent phosphorylation of CREB-1 and ATF-1.