Regulation of the mouse gene encoding TAFI by TNFα: role of NFκB binding site.
Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma pro-carboxypeptidase, encoded by the gene CPB2, with roles in both inhibition of fibrinolysis and inflammation. In mice, plasma TAFI levels and hepatic CPB2 mRNA expression were found to increase within 24h after intra-peritoneal lipopolysaccharide (LPS) injection. On the other hand, plasma TAFI in humans decrease in experimental endotoxemia and sepsis and we have previously demonstrated that CPB2 mRNA abundance in human hepatoma cells is decreased by inflammatory cytokines. Here, we have evaluated the effects of TNFα on mouse CPB2 expression. Treatment of primary mouse hepatocytes or the mouse hepatic cell line FL83B with TNFα for 12-48h resulted in increases in CPB2 mRNA abundance of up to 2-fold; mouse TAFI protein levels secreted from FL83B cells increased 2.7-fold after 48h treatment with TNFα. When FL83B cells were transfected with reporter plasmids containing the mouse CPB2 5'-flanking region, treatment with TNFα for 24 and 48h resulted in a 1.5-fold increased mouse CPB2 promoter activity. Mutation of a putative NFκB site not conserved in the human gene ablated the increased promoter activity observed following TNFα treatment. This site binds NFκB as assessed by gel mobility shift assays, and TNFα treatment increases the translocation of NFκB from the cytoplasm to the nucleus of mouse hepatocytes. These results demonstrate that the unique NFκB site in the mouse CPB2 promoter is functional and mediates the upregulation of mouse CPB2 expression by TNFα via increase in NFκB translocation to the nucleus.
Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.
Mathieu
M
Garand
Joellen H H
JH
Lin
Ceredwyn E
CE
Hill
Branislava
B
Zagorac
Marlys L
ML
Koschinsky
Michael B
MB
Boffa
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D025901:Carboxypeptidase B2 / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D025901:Carboxypeptidase B2