Regulation of the human chorionic gonadotropin alpha- and beta-subunit promoters by AP-2

Production of the placental hormone, chorionic gonadotropin (CG), increases dramatically as cytotrophoblasts fuse to form syncytiotrophoblasts. The CG alpha- and beta-promoters are both responsive to cAMP, although the kinetics of cAMP stimulation are different, In an effort to understand the kinetics of cAMP stimulation are different, In an effort to understand the mechanisms of coordinate induction of these genes, AP-2 binding sites were identified in the promoter regions of the alpha and CG beta genes, AP-2 bound to the upstream regulatory element (-186 to -156 base pairs (bp)) in the alpha-promoter and to several different regions of the CG beta promoter, including footprints 2 and 4B (FP2, -311 to -279 bp; FP4B, 221 to -200 bp). AP-2 antibodies induced supershifts of these complexes, confirming the identity of the protein-DNA complex. In JEG-3 cells, which contain abundant AP-2, mutations in these CG beta AP-2 sites reduced basal activity and decreased cAMP stimulation. In AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stimulated expression of the CG beta promoter 10-20-fold, and the alpha-promoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CG beta FP4B reduced AP-2 stimulation by more than 80%, whereas mutations in FP2 reduced AP-2 stimulation by less than 50%, Analyses of AP-2 mutants revealed a requirement for the DNA binding/dimerization do main and the amino terminal proline-rich and acid-rich transactivation domains for stimulation of the CGP promoter. Primary cultures of placental cytotrophoblasts were differentiated into syncytiotrophoblasts in vitro to examine AP-2 expression by reverse transcriptase-polymerase chain reaction. AP-2 mRNA levels increased by day 2 and continued to rise in parallel with a marked increase in alpha and CG beta gene expression. We conclude that both the alpha and CG beta promoters contain binding sites for AP-2 and suggest that this transcription factor provides a mechanism for coordinating the induction of these genes during placental cell differentiation.