Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level.

Abstract

Interleukin-6 (IL-6) is known to be a major mediator of the acute-phase response in liver. We show here that IL-6 triggers the rapid activation of a nuclear factor, termed acute-phase response factor (APRF), both in rat liver in vivo and in human hepatoma (HepG2) cells in vitro. APRF bound to IL-6 response elements in the 5'-flanking regions of various acute-phase protein genes (e.g., the %-macroglobulin, fibrinogen, and al-acid glycoprotein genes). These elements contain a characteristic hexanucleotide motif, CTGGGA, known to be required for the IL-6 responsiveness of these genes. Analysis of the binding specificity of APRF revealed that it is different from NF-IL6 and NF-KB, transcription factors known to be regulated by cytokines and involved in the transcriptional regulation of acute-phase protein genes. In HepG2 cells, activation of APRF was observed within minutes after stimulation with IL-6 or leukemia-inhibitory factor and did not require ongoing protein synthesis. Therefore, a preexisting inactive form of APRF is activated by a posttranslational mechanism. We present evidence that this activation occurs in the cytoplasm and that a phosphorylation is involved. These results lead to the conclusions that APRF is an immediate target of the IL-6 signalling cascade and is likely to play a central role in the transcriptional regulation of many IL-6-induced genes.During an acute inflammation, cytokines released by different cell types, including monocytes, fibroblasts, and endothelial cells, stimulate the synthesis and secretion of a set of plasma proteins, the so-called acute-phase proteins, by the liver. These proteins play a protective role during the acute-phase reaction, e.g., by inactivating proteases, supporting the wound-healing process, or scavenging free oxygen radicals (for a review, see reference 26). According to their regulation by different cytokines, acute-phase proteins have been divided into two subclasses (8). The synthesis of class 1 acute-phase proteins (e.g., a1-acid glycoprotein, C-reactive protein, haptoglobin, and serum amyloid A) is induced by interleukin-1 (IL-1) or combinations of IL-1 and IL-6, whereas the genes for class 2 acute-phase proteins (e.g., a2-macroglobulin, cxl-antichymotrypsin, and fibrinogen) are regulated mainly by IL-6 and glucocorticoids. The 5'-flanking regions of many acute-phase protein genes have been studied in detail, with the goal of identifying regulatory elements required for the cytokine induction of these genes. One type of cytokine response elements found in the promoters of several class 1 acute-phase protein genes represents binding sites for members of the C/EBP family of transcription factors (15,21,49,51,62 (36). In addition, NF-iB or NF-KB-like factors were found to be involved in the regulation of the angiotensinogen, serum amyloid A, and complement factor B genes by 48,54).Less is known about transcription factors regulating the promoters of class 2 acute-phase protein genes. On the basis of a sequence comparison of the 5'-flanking regions of the...

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

“…Previous several studies identified a highly conserved CTGG(G/A)AA motif in the IL-6-responsive regions of all three fibrinogen genes (10,33,34). This CTGG(G/ A)AA motif was also identified in the IL-6-responsive promoter region of the ␣ 2 -macroglobulin gene (35). Since the discovery of STAT3/APRF (36, 37), which interacts with a consensus element of TT(A/C)(C/T)N(G/A)(G/T)AA sequence, there has been intense effort to verify the role of the putative CTGG(G/A)AA motif and STAT3 in regulating the three fibrinogen genes.…”

Interleukin‐6‐induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin‐6 target gene induction

“…Nuclear proteins of HepG2 cells were extracted as previously described [14]. A double-stranded synthetic oligonucleotide comprising the IL-6RE of the ICAM-1 promoter (upper strand: 5'-AGCTTAG-GTTTCCGGGAAAGCAC-3') was 32p-labelled by filling in the 5'-protruding ends with Klenow enzyme.…”

“…We have previously identified a ubiquitous transcription factor, APRF, which, in response to IL-6, transiently associates with gpl30 and then is tyrosine phosphorylated and translocated to the nucleus [12,14]. On the basis of immunological cross-reactivity and DNA-binding specificities we proposed a relationship of APRF to the interferon-),-activated transcription factor Stat 1 [15].…”

“…Thus, APRF, now also called Stat 3, belongs to a growing family (the Stat family) of signal transducing and transactivating proteins which all are recruited by tyrosine phosphorylation from latent cytoplasmic forms in response to cytokines, hormones, and growth factors [16,[18][19][20][21]. In the nucleus, APRF bnds to IL-6 response elements (IL-6REs) in the flanking regions of many acute-phase protein genes [14]. Furthermore, we could demonstrate its binding to IL-6REs in the promoters of the immediate-early genes IRF-1, ICAM-I, and junB [22,23].…”

Role of STAT3 in inflammatory bowel disease

“…STAT3 was initially identified as an acute phase response factor, an inducible DNA binding protein that binds to the IL-6 responsive element within the promoters of hepatic acute phase protein genes [13] . Subsequent studies indicate that STAT3 becomes activated in response to a wide variety of cytokines and growth factors [4,5,14] .…”