ANALYSIS OF THE MECHANISM OF GLUCOCORTICOID-MEDIATED DOWN-REGULATION OF THE MOUSE ALPHA-FETOPROTEIN GENE

Regulation of alpha-fetoprotein gene expression by dexamethasone was e xamined in vivo and in vitro using primary mouse fetal liver cell cult ures. Dexamethasone accelerates the developmental down regulation of A FP mRNA pools. However, treatment of primary fetal liver cells in cult ure does not reduce the AFP mRNA pool and may stabilize both AFP and a lbumin 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 wa s examined by DNase I footprinting and transient expression assays. Tw o protein-binding regions of the proximal promoter (III and IV) show s ignificant homology to the GRE consensus sequence. DNase I footprintin g 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 adrenalectomiz ed mice show the same protection as controls. These results indicate t hat GR may not bind to the AFP proximal promoter in the adult. AFP pro moter-CAT expression vectors were used to further examine the effect o f dexamethasone on AFP expression. AFP promoter-CAT constructs were in hibited by 10(-6) M dexamethasone; while linking of an AFP enhancer to the promoter abolished the effect. We conclude that the in vitro effe cts 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.