A Novel cis-Acting Element Is Essential for Cytokine-Mediated Transcriptional Induction of the Serum Amyloid A Gene in Nonhepatic Cells

Abstract

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.

A SAF Binding Site in the Promoter Region of Human γ-Fibrinogen Gene Functions as an IL-6 Response Element

“…One member of this family, SAF-1, is homologous to human MAZ (28) or mouse Pur-1 (29). The broad array of other target genes that this family of proteins regulate are SAA (17)(18)(19)(20)(21), c-myc (28), insulin (29), serotonin 1A receptor (30), and the CD4 receptor (31). SAF-1 protein is inflammation responsive and is activated in several cell types, including the liver.…”

“…SAF-1 protein is inflammation responsive and is activated in several cell types, including the liver. LPS, IL-6, and minimally modified low density lipoprotein particles are a few among the known activating agents of SAF-1 (17)(18)(19)(20)(21). However, because SAF-1 is not expressed exclusively in the liver, liver-specific induction of the ␥-fibrinogen gene may not be mediated solely by this factor and may require some assistance.…”

“…Nuclear extract was prepared from uninduced and IL-6-induced cells as described (17). Protein content was measured by the Bradford method (25).…”

“…32 P-labeled double-stranded DNA probes for EMSA were prepared by filling in the overhangs at the termini with Klenow and [␣-32 P]dCTP. In EMSA, equal protein amounts of nuclear extracts were incubated with the radiolabeled DNA probe, and the resulting incubation products were electrophoresed in a nondenaturing 6% polyacrylamide gel as described (17). In some reactions, competitor oligonucleotides were included.…”

Functional Analysis of Interleukin 6 Response Elements (IL-6REs) on the Human γ-Fibrinogen Promoter

“…Therefore, we examined whether there might be additional IL-6-responsive elements on the human ␥FBG promoter by analysis of the promoter region up to Ϫ1000 bp from the ϩ1 site of transcription initiation. By computerassisted analysis, we looked for the type II IL-6 consensus motif CTGG(G)AA found in most Group 2 APP genes (30) and the SAF-1 consensus sequence RGGGRAGGRR where R represents any purine (31). In addition to the functional type II IL-6REs previously identified (13), two more putative type II IL-6REs were found.…”