IDENTIFICATION AND CHARACTERIZATION OF 2 PROMOTERS OF RAT KALLIKREIN-BINDING PROTEIN GENE

Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor (serpin) which binds to and inhibits tissue kallikrein activity [1,2] . In this study, we have sequenced and identified two promoter regions of the RKBP gene (RKBP). One promoter is located in the 5' flanking r egion (P-1) of the gene and the other is located in the first intron ( P-2). Both promoters contain a consensus TATA and CAAT box. These RKBP promoters were fused with a chloramphenicol acetyltransferase (CAT) r eporter gene and their promoter activities were determined by measurin g CAT levels using a specific ELISA. The P-1 promoter exhibited high p romoter activities in Hep3B hepatoma cells but not in La-fibroblastoma cells, indicating its tissue-specificity. By deletion analysis, we ha ve identified a negative regulatory element of the P-1 promoter betwee n -739 and -472, and defined a minimal sequence between -183 and -2 fo r maintaining the intact promoter activity, The P-2 promoter showed a strong activity only when linked to an SV40 enhancer. Activity of the P-1 promoter can be induced by growth hormone in Hep3B cells. Gel reta rdation assay has identified 5 DNA fragments which were bound by nucle ar proteins from rat liver. Two DNA fragments are in the 5' flanking r egion, one contains a putative glucocorticoid and growth hormone respo nse element and the other one contains a CAAT box and two putative AP- 1 binding sites. The remaining three are in the first intron and conta in a putative thyroid hormone response element, a putative GATA site a nd three consensus CAAT boxes, respectively. Nuclear proteins from the kidney showed that spontaneously hypertensive rats (SHR) have a disti nct trans-acting factor which binds with the DNA fragment containing t he glucocorticoid and growth hormone response elements, as compared wi th normotensive rats. This result indicates that different trans-actin g factors in the kidney of SHR may contribute to the decreased RKBP ex pression in these hypertensive rats.