Analysis of the mechanism of glucocorticoid-mediated down regulation of the mouse α-fetoprotein gene

Abstract

Regulation of α-fetoprotein gene expression by dexamethasone was examined in vivo and in vitro using primary mouse fetal liver cell cultures. Dexamethasone accelerates the developmental down regulation of AFP mRNA pools. However, treatment of primary fetal liver cells in culture does not reduce the AFP mRNA pool and may stabilize both AFP and albumin gene expression. These results indicate that in vivo the effect of dexamethasone may require interaction with another tissue or cell type. The mechanism of the dexamethasone mediated inhibition of AFP was examined by DNase I footprinting and transient expression assays. Two protein-binding regions of the proximal promoter (III and IV) show significant homology to the GRE consensus sequence. DNase I footprinting shows that only region IV can bind purified GR and competition with GRE oligonucleotides indicate that, using adult liver nuclear proteins, no GR is bound in either region. Nuclear protein from adrenalectomized mice show the same protection as controls. These results indicate that GR may not bind to the AFP proximal promoter in the adult. AFP promoter-CAT expression vectors were used to further examine the effect of dexamethasone on AFP expression. AFP promoter-CAT constructs were inhibited by 10 −6 M dexamethasone; while linking of an AFP enhancer to the promoter abolished the effect. We conclude that the in vitro effects on transiently expressed AFP directed expression vectors may be a function of vector structure and/or characteristics of the cells used whereas the in vivo effect may reflect normal regulatory mechanisms.