Interleukin-6 responsiveness and cell-specific expression of the rat kininogen gene.
The serum concentration of rat T1 kininogen increases 20-30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. To analyze the cis-regulatory elements responsible for the induced transcription, we fused a 1.6-kilobase segment of the rat T1 kininogen promoter to a reporter gene, chloramphenicol acetyltransferase (CAT). The resultant chimeric DNA was transfected into cultured cells. In transient transfection assays, this 5'-flanking sequence was sufficient to confer cell-specific expression: CAT activity was readily detectable when the construct was transfected into liver-derived cells, but it was not detectable in nonliver cells. Furthermore, when liver cells (Hep3B) transfected with this construct were treated with conditioned medium prepared from activated mixed lymphocyte cultures or with recombinant interleukin-6 (IL-6), a 5-fold increase in CAT activity was detected. Addition of dexamethasone to the conditioned medium or to IL-6 showed synergistic effects and resulted in a 10-fold increase in CAT activity. In contrast, when IL-1 was used with IL-6, induction of CAT activity was inhibited. Deletion analyses revealed two regions important for tissue-specific and induced regulation of T1 kininogen: sequences proximal to base pair -73 conferred enhanced expression in liver-derived cells and a distal region that conferred responsiveness to conditioned medium, recombinant IL-6, and dexamethasone. This responsive element had properties of an inducible transcriptional enhancer, and it was functional in both liver and nonliver cells when placed immediately upstream of a thymidine kinase promoter.
Chen
H M
HM
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Considine
K B
KB
Liao
W S
WS
D000818:Animals; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D007704:Kininogens / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D005938:Glucocorticoids; D015850:Interleukin-6; D007704:Kininogens; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase