Involvement of AP-1 and steroidogenic factor (SF)-1 in the cAMP-dependent induction of human adrenocorticotropic hormone receptor (ACTHR) promoter

Adrenocorticotropic hormone receptor (ACTHR) is expressed predominantly in the adrenal glands, and its expression is upregulated by its own ligand, AC TH, via a cAMP-dependent pathway. In the present study, we characterized th e 5'-regulatory region of human ACTHR gene to elucidate the molecular mecha nisms underlying its adrenal-specific and ACTH/cAMP-dependent expression. T he promoter region (-1017/+47 when the transcription start site is regarded as +1) and its serial 5'-deletions (-764/+47, -503/+47, -214/+47 and -56/47) were ligated into the upstream of a luciferase (luc) reporter gene. The se constructs were transfected into adrenocortical Y1 cells or non-adrenal JEG3 and Cos-1 cells. In all the cell lines, the luc activity gradually inc reased with serial 5'-deletions and the maximum activity was conferred by - 56/+47. However, the magnitude of luc activity of each deletion construct i n non-adrenal cells was much less than that in Y1 cells, suggesting that th e promoter functions in an adrenal-specific manner. We identified two Stero idogenic Factor (SF)-1-binding sites at -209 and -35. Electrophoretic mobil ity shift assay (EMSA) demonstrated that both sites bind to SF-1. Mutation of both sites significantly decreased the activity of -214/+47 promoter in Y1 cells. Transfection of SF-1-expressing plasmid into non-adrenal cells si gnificantly increased the promoter activity, suggesting that SF-1 plays a r ole in the tissue-specific expression of human ACTHR gene. We identified th e region, -764 to -503, that was required for the forskolin/cAMP responsive ness of the promoter. This region contains one AP-1 site. EMSA revealed tha t the binding of AP-1 to this site increased significantly upon treatment o f Y1 cells with forskolin. Mutation of the site abolished the forskolin-res ponsiveness. In non-adrenal cells, the forskolin-responsiveness was observe d only when SF-1-expressing plasmid was cotransfected. This is the first de monstration that both AP-1 and SF-1 are required for the cAMP-dependent ind uction of human ACTHR gene.