Ad4BP/SF-1 Regulates Cyclic AMP-induced Transcription from the Proximal Promoter (PII) of the Human Aromatase P450 (CYP19) Gene in the Ovary

Abstract

Aromatase P450, which is responsible for the metabolism of C19 steroids to estrogens, is expressed in the pre-ovulatory follicles and corpora lutea of ovulatory women by means of a promoter proximal to the start of translation (PII). To understand how this transcription is controlled by cAMP, we constructed chimeric constructs containing deletion mutations of the proximal promoter 5′-flanking DNA fused to the rabbit β-globin reporter gene. Assay of reporter gene transcription in transfected bovine granulosa and luteal cells revealed that cAMP-stimulated transcription was lost upon deletion from −278 to −100 base pairs, indicating the presence of a functional cAMP-responsive element in this region; however, no classical cAMP-responsive element was found. Mutation of an AGGTCA motif located at −130 base pairs revealed that this element is crucial for cAMP-stimulated reporter gene transcription. When a single copy of this element was placed upstream of a heterologous promoter, it could act as a weak cAMP-response element. Supershift electrophoretic mobility shift assay and UV cross-linking established that Ad4BP/SF-1 binds to this hexameric element. Ad4BP/SF-1 mRNA and protein levels and DNA binding activity are increased in forskolin-treated luteal cells. We conclude that cAMP-stimulated transcription of human aromatase P450 in the ovary is due, at least in part, to increased levels and DNA binding activity of Ad4BP/SF-1. Aromatase P450, which is responsible for the metabolism of C19 steroids to estrogens, is expressed in the pre-ovulatory follicles and corpora lutea of ovulatory women by means of a promoter proximal to the start of translation (PII). To understand how this transcription is controlled by cAMP, we constructed chimeric constructs containing deletion mutations of the proximal promoter 5′-flanking DNA fused to the rabbit β-globin reporter gene. Assay of reporter gene transcription in transfected bovine granulosa and luteal cells revealed that cAMP-stimulated transcription was lost upon deletion from −278 to −100 base pairs, indicating the presence of a functional cAMP-responsive element in this region; however, no classical cAMP-responsive element was found. Mutation of an AGGTCA motif located at −130 base pairs revealed that this element is crucial for cAMP-stimulated reporter gene transcription. When a single copy of this element was placed upstream of a heterologous promoter, it could act as a weak cAMP-response element. Supershift electrophoretic mobility shift assay and UV cross-linking established that Ad4BP/SF-1 binds to this hexameric element. Ad4BP/SF-1 mRNA and protein levels and DNA binding activity are increased in forskolin-treated luteal cells. We conclude that cAMP-stimulated transcription of human aromatase P450 in the ovary is due, at least in part, to increased levels and DNA binding activity of Ad4BP/SF-1.