Glucocorticoid receptor binding site in the mouse alpha-amylase 2 gene mediates response to the hormone

Amylase gene expression has been shown to be positively regulated by glucocorticoids. Previous reports have suggested that this effect is indirect. We have addressed this question in a mouse exocrine pancreas cell line, 266-6, in which basal level of expression of amylase mRNA is low but inducible by glucocorticoids. In these cells the effect of glucocorticoids is not inhibited by cycloheximide at early time points. Reporter plasmids containing 224 base pairs of mouse amylase 5'-flanking DNA are positively regulated by glucocorticoids in gene transfer experiments. Glucocorticoid receptor purified from rat liver binds to the amylase promoter from position -56 to -33 and at the start of transcription. Site-directed mutation at the upstream position (-47 to -42) eliminates response to glucocorticoids in transient gene transfer experiments. Thus, glucocorticoid regulation of the mouse amylase gene is a direct effect and is mediated via a receptor binding site in the promoter region of the gene. Inhibition of the hormone response by cycloheximide at later time points after induction suggests the additional requirement for a short-lived factor. The DNA binding domain of the glucocorticoid receptor binds to a single site in the amylase promoter as a monomer, suggesting that both receptor binding sites as well as an additional short-lived factor are required to obtain induction.