Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene.

Abstract

Interleukin 6 (IL-6) was established as a transcriptional inducer of the rat a2-macroglobulin gene, a prototype liver acute-phase gene. Maximum induction occurred when the 5' flanking sequences of this gene (position -209 to -43) directed expression from the gene's own TATA box and transcription start site. Removal of the hexanucleotide CTGGGA (position -164 to -159) abolished 60-70% of the hormonal induction in FAO1 rat hepatoma cells. This hexanucleotide was dermed as the IL-6 response element (IL-6-RE).The IL-6-RE is well conserved in the cytokine-responsive regions of other acute-phase genes and serves as a binding site for nuclear proteins. A characteristic DNA-protein complex (complex I) was formed with nuclear proteins from normal rat livers. A different, hormone-inducible complex (complex II) was assembled specifically with nuclear proteins from acutephase rat livers or from IL-6-treated human Hep 3B hepatoma cells. Complex II was competitively inhibited by oligonucleotides representing the conserved IL-6-RE sequence from other acute-phase genes. Thus, the proteins building complex II likely participate in a general signal transduction mechanism mediating the transcriptional activation by IL-6 of several acute-phase genes.Interleukin 6 (IL-6) coordinates several important reactions in inflammation and the host defense against tissue damage and infections (1). It induces the synthesis of protective plasma proteins (acute-phase proteins) in the liver. It also modulates the immune response and regulates body temperature and fever (2-5). We have chosen the rat a2-macroglobulin (a2M) gene to study gene activation by because this gene is dramatically induced by IL-6. a2M contributes to the host defense as a broad-range proteinase inhibitor and as a carrier of various hormones (6-13). During an acute inflammatory reaction, a2M plasma concentrations rise several 100-fold within 48 hr, following a corresponding elevation of hepatic a2M mRNA levels (6,7,14). Transcription rates of the gene were reportedly increased up to 9-fold (6, 7, 15, 16). In living rats and most hepatic cell culture systems, both IL-6 and glucocorticoids are required for the full induction of the a2M gene (3,17,18). Here we have evaluated the respective contributions of IL-6 and glucocorticoids to the transcriptional induction of the a2M gene by using a hepatoma cell line stably transfected with an a2M gene promoter region construct. In this assay system, IL-6 alone induced transcription of the a2M gene, and glucocorticoids enhanced its effect. Using mutagenesis and transfection into hepatoma cells, we have mapped an IL-6-responsive cis-acting transcriptional control element in the 5' flanking region ofthe a2M gene. This element serves as a binding site for specific nuclear proteins, and we have observed a hormone-induced characteristic protein-DNA complex between this element and nuclear proteins from acute-phase rat livers and IL-6-treated cultured hepatoma cells. MATERIALS AND METHODSCell Cultures, DNA Transfections, and Luciferas...

A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.

“…A 100-fold excess of a fragment from the human albumin gene promoter (halb) was also successful in competing with the C3 fragment for formation of all complexes, suggesting common factor-binding sites Sequences similar to the IL--responsive element are found within the human C3 promoter. Hattori et al (20) and Ito et al (23) have identified the hexanucleotide sequence CTGGGA as being important in the IL-6 regulation of the acute-phase gene for a2-macroglobulin. This sequence is found in a number of other IL-6-responsive acute-phase genes (14,23,33).…”

“…4B), TCTGGRA, found in the regulatory regions defined for other acute-phase genes (14,33), including the haptoglobin and CRP genes. The functional importance of this element in the IL-6 induction of these two genes and of the a2-macroglobulin gene (20,23) has been established. We have replaced our CTGGGG hexanucleotide with an EcoRV site and have found it to cause a drastic change in both the base-line (unstimulated) and IL-1-inducible expression of the C3 gene.…”

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

“…The consensus recognition motif for the glucocorticoid response element (TGT(C/T)CT) (37) was represented 4 times at positions Ϫ701 to Ϫ696, Ϫ636 to Ϫ631, Ϫ442 to Ϫ437, and Ϫ66 to Ϫ61. Two interleukin-6 response elements (38), which act as positive elements in several acute phase protein genes, were identified at Ϫ1301 to Ϫ1296, in reverse complement form, and at Ϫ317 to Ϫ312. Two NF-1 binding motifs (5Ј-TGGN 7 CCA-3Ј) (39) were found at Ϫ1218 to Ϫ1206 and Ϫ630 to Ϫ618, the latter overlapping with a glucocorticoid response element.…”

STAT3 activation by cytokines utilizing gp130 and related transducers involves a secondary modification requiring an H7-sensitive kinase.

“…One of these is STAT1 (13,15,23), the DNA-binding transcriptional activator first identified for its role in interferon signaling (24,25). Recent work has revealed that IL-6, CNTF, and their relatives can activate a second molecularly cloned STAT protein (26,27), termed STAT3, which binds DNA response elements similar to those recognized by STAT1 and apparently corresponds to a DNA-binding activity previously referred to as acute-phase response factor (APRF) (28)(29)(30). The CNTF family also induces tyrosine phosphorylation of two closely migrating proteins designated p88 and p90 that, while similar in size to STAT1 and weakly immunoreactive with antibodies to STAT1, were shown not to correspond to STAT1 (13,15,23); we considered the possibility that at least one of these might correspond to STAT3.…”