Varied Mechanisms of Oestradiol-Mediated Regulation of Dopamine β-Hydroxylase Transcription

Abstract

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine β-hydroxylase (DBH) gene transcription. In the present study, we examined oestrogen-responsive elements of the rat DBH gene promoter aiming to characterise the mechanisms of oestradiol-induced DBH transcription. Various mutations and deletions of DBH promoter reporter constructs were tested for responsiveness to 17β-oestradiol (E(2) ). Mutation of the half palindromic oestrogen response element (ERE) at position -759 reduced the response to E(2) in PC12 cells co-transfected with oestrogen receptor (ER) α, indicating a functional role for this motif. In cells co-transfected with ERβ, mutations at the -759 site were unresponsive to E(2) . To characterise the additional E(2) responsive elements, mediated by ERα, the DBH promoter was truncated to the proximal 249 or 200 nucleotides upstream of the transcription start site. Despite either truncation, 10 nm E(2) still elicited an approximately two-fold induction of DBH promoter activity. Mutation of a possible ERE-like sequence at -59 had no effect. The lack of a functional ERE in the proximal region of the rat DBH promoter despite E(2) -mediated DBH promoter activity, suggests regulation by a nonclassical mechanism, such as a membrane-initiated signalling pathway. Moreover, the induction of DBH promoter activity and the rise in DBH mRNA levels were observed within hours. To determine whether membrane-initiated E(2) signalling is involved in rat DBH gene transcription, a membrane impermeable E(2) conjugate, β-oestradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin (E(2) BSA), was used. Incubation with E(2) -BSA induced luciferase activity and elicited a significant rise in DBH mRNA levels in the ERα transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E(2) in the presence of ERα. The results implicate both genomic and membrane-initiated mechanisms, mediated by ERα, in E(2) -induced DBH gene transcription.