MOLECULAR ANALYSIS OF THE DIFFERENTIAL HEPATIC EXPRESSION OF RAT KININOGEN FAMILY GENES

Serum concentration of rat TI kininogen increases 20- to 30-fold in re sponse to acute inflammation, an induced hepatic synthesis regulated p rimarily at the transcriptional level. We have demonstrated by transie nt transfection analyses that rat T1 kininogen gene/chloramphenicol ac etyltransferase (T1K/CAT) constructs are highly responsive to interleu kin-6 and dexamethasone. In these studies we examined the regulation o f a highly homologous K kininogen gene promoter and showed that it is minimally induced under identical conditions. The basal expression of the KK/CAT construct was, however, five- to sevenfold higher than that of the analogous T1K/CAT construct. Promoter-swapping experiments to examine the molecular basis of this differentially regulated basal exp ression showed that at least two K kininogen promoter regions are impo rtant for conferring its high basal expression: a distal 19-bp region (C box) constituted a binding site for C/EBP family proteins, and a pr oximal 66-bp region contained two adjacent binding sites for hepatocyt e nuclear factor 3 (HNF-3). While the C box in the K kininogen promote r was able to interact with C/EBP transcription factors, the T1 kinino gen promoter C box could not. In addition, HNF-3 binding sites of the K kininogen promoter demonstrated stronger affinities than those of th e T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and are known to enhance transcription of liver-specific gen es, these differences in their binding activities thus accounted for t he K kininogen gene's higher basal expression. Our studies demonstrate d that evolutionary divergence of a few critical nucleotides may lead to subtle changes in the binding affinities of a transcription factor to its recognition site, profoundly altering expression of the downstr eam gene.