Novel activator protein-2 alpha splice-variants function as transactivators of the ovine placental lactogen gene

Activator protein-2 (AP-2) has been implicated as a transactivator of the h uman and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expre ssed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected hum an choriocarcinoma cells (BeWo). AP-2 alpha was localized in ovine chorioni c epithelial cells by immunohistochemistry and a 3-kb transcript was identi fied by Northern hybridization. Four nearly full-length AP-2 cDNAs were iso lated from an ovine placenta cDNA library. Nucleotide sequencing these cDNA s revealed that the AP-2 mRNA expressed in the ovine placenta shares identi ty with human AP-2 alpha, but variations in the predicted N-terminus were o bserved, and three unique AP-2 alpha splice-variants were identified. Expre ssion of AP-2 alpha variants in HepG2 cells, devoid of endogenous AP-2, ind icates that enhancement through the AP-2 element in the oPL gene minimal pr omoter was variant dependent. RNA transcripts for all of the ovine AP-2 alp ha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2 alph a transcript, which shares identity to Xenopus AP-2 alpha, was expressed in BeWo cells. immunoblot analysis confirmed AP-2 alpha variants in ovine cho rionic binucleate cell nuclear extracts, one of which migrates similar to t he AP-2 alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2 alpha splice-variants that augm ent transactivation of the oPL gene in ovine chorionic binucleate cells.