title: Involvement of AP-1 and steroidogenic factor (SF)-1 in the cAMP-dependent induction of human adrenocorticotropic hormone receptor (ACTHR) promoter.
abstract: Adrenocorticotropic hormone receptor (ACTHR) is expressed predominantly in the adrenal glands, and its expression is upregulated by its own ligand, ACTH, via a cAMP-dependent pathway. In the present study, we characterized the 5'-regulatory region of human ACTHR gene to elucidate the molecular mechanisms underlying its adrenal-specific and ACTH/cAMP-dependent expression. The 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. These constructs were transfected into adrenocortical Y1 cells or non-adrenal JEG3 and Cos-1 cells. In all the cell lines, the luc activity gradually increased with serial 5'-deletions and the maximum activity was conferred by - 56/+ 47. However, the magnitude of luc activity of each deletion construct in non-adrenal cells was much less than that in Y1 cells, suggesting that the promoter functions in an adrenal-specific manner. We identified two Steroidogenic Factor (SF)-1-binding sites at -209 and -35. Electrophoretic mobility 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 significantly increased the promoter activity, suggesting that SF-1 plays a role in the tissue-specific expression of human ACTHR gene. We identified the region, -764 to -503, that was required for the forskolin/cAMP responsiveness of the promoter. This region contains one AP-1 site. EMSA revealed that the binding of AP-1 to this site increased significantly upon treatment of Y1 cells with forskolin. Mutation of the site abolished the forskolin-responsiveness. In non-adrenal cells, the forskolin-responsiveness was observed only when SF-1-expressing plasmid was cotransfected. This is the first demonstration that both AP-1 and SF-1 are required for the cAMP-dependent induction of human ACTHR gene.
authors.affiliation: Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Sarkar
authors.forename: F
authors.initials: F
authors.lastname: Kambe
authors.forename: Y
authors.initials: Y
authors.lastname: Hayashi
authors.forename: S
authors.initials: S
authors.lastname: Ohmori
authors.forename: H
authors.initials: H
authors.lastname: Funahashi
authors.forename: H
authors.initials: H
authors.lastname: Seo
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004586:Electrophoresis; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D018043:Receptors, Corticotropin / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D018043:Receptors, Corticotropin; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D000242:Cyclic AMP