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

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine beta-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 17 beta-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) alpha, indicating a functional role for this motif. In cells co-transfected with ER beta, mutations at the -759 site were unresponsive to E-2. To characterise the additional E-2 responsive elements, mediated by ER alpha, 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, beta-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 ER alpha transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E-2 in the presence of ER alpha. The results implicate both genomic and membrane-initiated mechanisms, mediated by ER alpha, in E-2-induced DBH gene transcription.