Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-8 and C/EBP-0 are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-8 or C/EBP-0 confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-8 isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-8 prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-8 is a phosphoprotein. These results suggest that C/EBP-8 is regulated by phosphorylation and, in conjunction with C/EBP-P, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.

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The promoter region of the rabbit serum amyloid A (SAA) gene contains two adjacent C/EBP and one NF-kappa B binding element. Involvement of these elements in SAA gene induction, following lipopolysaccharide (LPS) stimulation of the liver, has been studied by investigating LPS-activated transcription factors and their interaction with the promoter elements of the SAA gene. Appearance of complexes in the electrophoretic mobility shift assay has indicated that DNA-binding proteins that interact with the NF-kappa B element of the SAA promoter are induced in the LPS-treated rabbit liver. Presence of RelA (p65 subunit of NF-kappa B) in these complexes was demonstrated by the ability of RelA-specific antisera to supershift the DNA-protein complexes. LPS also induced several members of the C/EBP family of transcription factors, which interacted with the C/EBP motifs of the SAA promoter. Activated C/EBP and RelA form a RelA-C/EBP heteromeric complex that associates with varying affinity to NF-kappa B and C/EBP elements of the SAA gene. Transfection assays using both transcription factor genes have demonstrated that the heteromeric complex of NF-kappa B and C/EBP is a much more potent transactivator of SAA expression than each transcription factor alone. The heteromeric complex efficiently promotes transcription from both NF-kappa B and C/EBP sites.

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between ؊254 and ؊226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6.

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions ؊280 and ؊224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of a protein phosphatase inhibitor, and loss of IL-6-mediated inducible DNA-binding activity and reporter gene activation in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.