Oestradiol decreases rat apolipoprotein AI transcription via promoter site B

Abstract

Oestrogens protect against ischaemic heart disease in the post-menopausal female by increasing serum concentrations of apolipoprotein (apo) AI and the abundance of high-density lipoprotein particles. In men and experimental male animals, the administration of oestrogen has variable effects on apo AI expression. As the major mode of oestrogen action on target genes involves regulating promoter activity and hence transcription, oestrogen is expected to alter transcription of the apo AI gene. To test this hypothesis, the effect of 17β-oestradiol (E2), on rat apo AI promoter activity in male hepatoma HUH-7 cells, was tested by cotransfecting a reporter template, pAI.474.CAT containing -474 to -7 of the rat apo AI promoter and an oestrogen receptor (ER) expression vector, pCMV-ER. Transfected cells exposed to E2 showed a dose-dependent decrease in chloramphenicol acetyltransferase (CAT)-activity, with a maximum 91 ± 1.5% reduction at 1 μM E2. Deletional analysis of the promoter localized the inhibitory effect of ER and E2 to site B (-170 to -144) with an adjacent 5' contiguous motif, site S (-186 to -171) acting as an amplifier. HuH-7 cell nuclear extracts showed binding activities with both sites S and B, but recombinant human ER did not. Furthermore, nuclear extracts from E2-treated HuH-7 cells showed weaker binding activity to site B, but not to site S. In summary, the inhibitory effect of ER and E2 on rat ado AI gene activity is mediated by a promoter element, site B. This inhibitory effect arises from a mechanism that does not involve direct ER binding to the B-element. The conclusion that E2 inhibits apo AI transcription was confirmed in vivo. Treatment of male adult Sprague-Dawley rats with up to 200 μg E2 for 7 days decreased apo AI protein and hepatic mRNA by 724±21% and 68 ± 1.4% respectively. Results of 'run-on' transcription of the apo AI gene in isolated hepatic nuclei showed a 55% decrease in hormone-treated male rats. These findings suggest that E2 exerts primarily an inhibitory effect within male hepatic nuclei.