Identification and characterization of two 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 region (P1) of the gene and the other is located in the first intron (P2). Both promoters contain a consensus TATA and CAAT box. These RKBP promoters were fused with a chloramphenicol acetyltransferase (CAT) reporter gene and their promoter activities were determined by measuring CAT levels using a specific ELISA. The P1 promoter exhibited high promoter activities in Hep3B hepatoma cells but not in La-fibroblastoma cells, indicating its tissue-specificity. By deletion analysis, we have identified a negative regulatory element of the P1 promoter between -739 and -472, and defined a minimal sequence between -183 and -2 for maintaining the intact promoter activity. The P2 promoter showed a strong activity only when linked to an SV40 enhancer. Activity of the P1 promoter can be induced by growth hormone in Hep3B cells. Gel retardation assay has identified 5 DNA fragments which were bound by nuclear proteins from rat liver. Two DNA fragments are in the 5' flanking region, one contains a putative glucocorticoid and growth hormone response 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 contain a putative thyroid hormone response element, a putative GATA site and three consensus CAAT boxes, respectively. Nuclear proteins from the kidney showed that spontaneously hypertensive rats (SHR) have a distinct trans-acting factor which binds with the DNA fragment containing the glucocorticoid and growth hormone response elements, as compared with normotensive rats. This result indicates that different trans-acting factors in the kidney of SHR may contribute to the decreased RKBP expression in these hypertensive rats.