Nucleic Acids Research

We have examined the regulation of a rat T-kininogen gene by glucocorticoid and estrogen. Expression of the endogenous gene in a rat hepatoma cell line is increased 5fold and 2-fold in response to dexamethasone and 17B-estradiol-3-benzoate, respectively. Various deletion constructs of the 5 region of an isolated T-kininogen gene were fused to a ehloramphenicol acetyltransferase (CAT) gene and introduced into the hepatoma cells by electroporation. Analysis of the CAT activity in cell extracts after treatment with glucocorticoid or estrogen revealed that a fragment from -167 to +52 is sufficient to confer full induction. An additional deletion in this region was unresponsive, while a larger fragment (-612 to -100) linked to a heterologous promoter did result in regulated expression. These results suggested that the sequence responsible for the hormonal response was located at -167 to -100 from the transcription start site. This 67 bp region contains a consensus for the core sequence of the glucocorticoid responsive element (GRE) and the estrogen responsive element (ERE). Interestingly these elements are located within 7 bp of each other and both sequences overlap a 16 bp palindrome that may be important in hormone receptor-DNA recognition. INTRODUCTION Tissue injury brought about by bacterial infections, burns, or malignant growths triggers a systemic inflammatory response in mammals. One aspect of this response is increased activity of the adrenal gland and a concomitant rise in the concentration of circulating glucocorticoid hormone (1). Furthermore, there is a change in the profile of serum proteins produced by the liver. Albumin and transferrin levels are decreased, while a range of other proteins are dramatically increased. As a group, these proteins have been referred to as acute phase inflammatory reactants (2). In rats, a cysteine protease inhibitor is induced 8-10 fold in response to inflammation (3,4). It has been called al-major acute phase protein to reflect this induction (5). More recently, it is referred to as T-kininogen (T-KG) because of the high degree of sequence similarity to the low molecular weight K-kininogen (6,7). Tkininogen contains a bradykinin moeity as do the K-kininogens, however the amino acids flanking this nonapeptide are different in T-kininogen. As a result, T-kininogen will only serve as precursor of bradykinin if the protein is treated with excess trypsin (8). The