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 g lucocorticoids. Previous reports have suggested that this effect is in direct. We have addressed this question in a mouse exocrine pancreas c ell line, 266-6, in which basal level of expression of amylase mRNA is low but inducible by glucocorticoids. In these cells the effect of gl ucocorticoids is not inhibited by cycloheximide at early time points. Reporter plasmids containing 224 base pairs of mouse amylase 5'-flanki ng DNA are positively regulated by glucocorticoids in gene transfer ex periments. Glucocorticoid receptor purified from rat liver binds to th e amylase promoter from position -56 to -33 and at the start of transc ription. Site-directed mutation at the upstream position (-47 to -42) eliminates response to glucocorticoids in transient gene transfer expe riments. Thus, glucocorticoid regulation of the mouse amylase gene is a direct effect and is mediated via a receptor binding site in the pro moter region of the gene. Inhibition of the hormone response by cycloh eximide at later time points after induction suggests the additional r equirement for a short-lived factor. The DNA binding domain of the glu cocorticoid 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.