Identification of a glucocorticoid response element contributing to the constitutive expression of the rat liver alpha 1-inhibitor III gene.

alpha 1-Inhibitor III (alpha 1 I3), a broad range plasma proteinase inhibitor, is synthesized with striking tissue specificity in rat livers. The gene is expressed strongly in periportal hepatocytes of healthy adults and less abundantly in regions near the centrilobular vein. This expression pattern is suggestive of a concentration gradient of a blood-borne hormone that enters through the portal vein and diffuses across the lobe toward the centrilobular vein. The alpha 1 I3 gene was known to be regulated both by glucocorticoids and interleukin 6, and therefore the hypothesis was tested that the normal constitutive expression of this gene depended on glucocorticoids. alpha 1 I3 mRNA levels in the livers of hypophysectomized rats with low endogenous glucocorticoid levels were only about 20% of those in control rat livers. Injection of exogenous glucocorticoids reconstituted hepatic alpha 1 I3 mRNA levels up to 64% of their original values in a dose-dependent manner. Similarly, treatment of FAZA rat hepatoma cells with the synthetic glucocorticoid dexamethasone induced alpha 1 I3 mRNA levels in a dose-dependent manner. Taken together these data suggested that glucocorticoids are required for the constitutive high level expression of this gene in normal adult rat livers. A series of 5' deletion constructs and linker scanning mutants of the promoter upstream region were produced and transfected into FAZA cells. A functional glucocorticoid response element was mapped between -168 and -151 base pairs 5' of the transcription start site. This element conforms with an inverted consensus glucocorticoid response element (GRE) but differs in two positions essential for protein DNA interaction between the GRE and the glucocorticoid receptor (GR). The induction of alpha 1 I3 gene promoter region constructs by dexamethasone was abolished by the receptor antagonist RU486, indicating that the GR participated in the activation of the alpha 1 I3 gene. In DNase I footprinting experiments with nuclear protein extracts from untreated and dexamethasone-treated FAZA cells, similar extents of alpha 1 I3 promoter upstream sequences were protected, indicating that proteins capable of binding in the glucocorticoid response-mediating element (GME) region were present before and after arrival of the hormonal signal. However, a purified recombinant fragment of the GR which contained essentially only its DNA binding domain was unable to bind at the GME although it interacted strongly with a consensus GRE sequence.