Cyclic AMP- and differentiation-dependent regulation of the proximal alphaHCG gene promoter in term villous trophoblasts

Although the regulatory mechanisms controlling alpha and beta human chorion ic gonadotrophin (HCG) expression have been investigated in choriocarcinoma cell model systems, little is known about the regulation of HCG subunit sy nthesis in non-tumourigenic trophoblasts. We therefore investigated alpha H CG mRNA transcription in villous cytotrophoblasts isolated from term placen tae and have shown for the first time that the proximal alpha HCG gene prom oter is functional in these cells. By establishing conditions which allow e fficient transient transfection of immunopurified cells, we have demonstrat ed that a 363 bp sequence in the proximal 5' flanking region of the alpha H CG gene is sufficient to direct trophoblast-specific expression of a lucife rase reporter. After 12-60 h cultivation, an increase in endogenous alpha H CG mRNA expression could be detected, indicating that aggregated villous tr ophoblasts undergo biochemical differentiation. Concomitantly, we observed induction of alpha HCG promoter-driven luciferase activity, suggesting that the 363 bp sequence of the proximal 5' flanking region is sufficient to di rect differentiation-dependent increase of alpha HCG mRNA. Continuous lucif erase expression required functional cAMP-response elements (CREs), since d eletion of both recognition sequences eliminated differentiation-dependent transcription of the reporter. Elevation of cAMP values increased transcrip tion of the wild-type construct; however, it did not affect promoter activi ty of the mutant plasmid. Moreover, we have demonstrated that during in-vit ro differentiation, CREs interacted with increasing amounts of phosphorylat ed activating transcription factor/cyclic AMP response element-binding prot ein (ATF-1/CREB-1) suggesting that these cAMP-dependent DNA-binding factors are major determinants in regulating alpha HCG gene expression in villous trophoblasts.