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G H Fey

Publications and source records attributed to G H Fey.

At least 19 recordsLinked to original sources
PubMed1992

Expression of a functional alpha-macroglobulin receptor binding domain in Escherichia coli.

We have expressed receptor-binding domains of human alpha 2-macroglobulin and rat alpha 1-macroglobulin in Escherichia coli. Expression levels of both recombinants were quite high, but the human one was insoluble, probably forming inclusion bodies. The rat domain, which lacks the human disulfide, was produced in a soluble form and readily purified by two simple chromatographic steps. Purified recombinant rat alpha 1-macroglobulin receptor-binding domain was fully functional in binding to the alpha-macroglobulin receptor on human fibroblasts. This 142 residue domain should serve as an excellent template for analyzing the structural requirements for alpha-macroglobulin receptor ligation and dissecting the varied biological functions resulting from such ligation.

Animals↗
PubMed1992

Synergistic action of interleukin-6 and glucocorticoids is mediated by the interleukin-6 response element of the rat alpha 2 macroglobulin gene.

One class of genes coding for the acute-phase proteins (acute-phase genes) is induced by interleukin 6 (IL-6) through the human transcription factor NF-IL-6 and its rat homolog IL-6-DBP/LAP. A second class, represented by the rat alpha 2 macroglobulin gene, utilizes a different IL-6 response element (IL-6-RE) and different DNA-binding proteins interacting with this element, the so-called IL-6-RE binding proteins (IL-6 RE-BPs). Human Hep3B and HepG2 hepatoma, U266 myeloma, and CESS lymphoblastoid cells contain IL-6 RE-BPs that form complexes, with the IL-6-RE, with gel mobilities indistinguishable from those of the corresponding complexes of rat liver cells. The ability to form these complexes was induced by IL-6 in human hepatoma cells with a maximum reached after 4 h and required ongoing protein synthesis. Multiple copies of an 18-bp element containing the IL-6-RE core were sufficient to confer both induction by IL-6 and a synergistic induction by IL-6 plus glucocorticoids to minimal promoters. The synergism was blocked by the receptor antagonist RU486 and thus was dependent on the glucocorticoid receptor (GR). However, the 18-bp element contained no consensus GR-binding site, and recombinant GR did not bind at this sequence. Therefore, the synergism was probably achieved by an indirect effect of a glucocorticoid-activated intermediate gene on the IL-6 RE-BPs. The rat IL-6 RE-BP had a molecular weight of 102 +/- 10 kDa and was thus distinct from NF-IL-6 and IL-6-DBP/LAP. Therefore, IL-6 must activate two different classes of liver acute-phase genes through at least two different nuclear DNA-binding proteins: NF-IL-6/IL-6-DBP/LAP and the IL-6 RE-BP.

Animals↗
PubMed1992

Alpha 2-macroglobulin expression in the mesometrial decidua and its regulation by decidual luteotropin and prolactin.

During decidualization, cells of the endometrium grow and differentiate giving rise to two different decidual tissues located in either the antimesometrial or mesometrial site of the uterus in the rat. These tissues have different functions in pregnancy. The antimesometrial decidua is an endocrine gland that secretes hormones, whereas the mesometrial decidua appears to play an important role in limiting trophoblast invasion. Since the decidual tissue of the rat produces alpha 2-macroglobulin (alpha 2MG), we examined whether this potent protease inhibitor is specifically expressed by the mesometrial tissue, the site of trophoblast invasion, and whether the alph 2MG gene is regulated by decidual luteotropin (DLt), the PRL-like hormone secreted by the neighboring antimesometrial cells. To determine the secretory proteins of the rat decidua, antimesometrial and mesometrial tissues were dissected out from pseudopregnant rats and cultured with [35S]methionine. The major protein secreted by the antimesometrial cell was the 29-kilodalton decidual luteotropin, whereas a 180-kilodalton protein was predominantly secreted by the mesometrial tissue. Immunoprecipitation studies of 35S-radiolabeled proteins revealed that this high mol wt protein is alpha 2MG and that it is secreted exclusively by the cells forming the mesometrial tissue. To examine whether the alpha 2MG gene was also expressed specifically in the mesometrial decidua, total RNA was isolated from both mesometrial and antimesometrial tissues of day 9-12 pseudopregnant rats and hybridized with alpha 2MG cDNA. Northern blot analysis revealed a 5.4-kilobase message, which was abundantly expressed in the mesometrial decidua. Little, if any, alpha 2MG mRNA was detected in antimesometrial decidua. The ontogeny of the message in the decidua correlated well with the development of the mesometrial tissue. To examine whether alpha 2MG expression is regulated by DLt and/or PRL, a highly specific polyclonal antibody to DLt was generated and decidual tissues were cultured in the presence or absence of DLt antibodies with or without PRL. Neutralization of DLt caused a marked decrease in alpha 2MG mRNA levels. This down-regulation was totally reversed by the addition of PRL and was not affected by alpha 2MG antibodies. In summary, the results of this investigation revealed a compartmentalized gene expression, synthesis and secretion of alpha 2MG in the decidua. The secretion of this protease inhibitor, specifically by the mesometrial tissue which is the site of trophoblast invasion, may be the reason for the minimal amount of tissue damage that occurs during placentation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Identification of a glucocorticoid response element contributing to the constitutive expression of the rat liver alpha 1-inhibitor III gene.

alpha 1-Inhibitor III (alpha 1 I3), a broad range plasma proteinase inhibitor, is synthesized with striking tissue specificity in rat livers. The gene is expressed strongly in periportal hepatocytes of healthy adults and less abundantly in regions near the centrilobular vein. This expression pattern is suggestive of a concentration gradient of a blood-borne hormone that enters through the portal vein and diffuses across the lobe toward the centrilobular vein. The alpha 1 I3 gene was known to be regulated both by glucocorticoids and interleukin 6, and therefore the hypothesis was tested that the normal constitutive expression of this gene depended on glucocorticoids. alpha 1 I3 mRNA levels in the livers of hypophysectomized rats with low endogenous glucocorticoid levels were only about 20% of those in control rat livers. Injection of exogenous glucocorticoids reconstituted hepatic alpha 1 I3 mRNA levels up to 64% of their original values in a dose-dependent manner. Similarly, treatment of FAZA rat hepatoma cells with the synthetic glucocorticoid dexamethasone induced alpha 1 I3 mRNA levels in a dose-dependent manner. Taken together these data suggested that glucocorticoids are required for the constitutive high level expression of this gene in normal adult rat livers. A series of 5' deletion constructs and linker scanning mutants of the promoter upstream region were produced and transfected into FAZA cells. A functional glucocorticoid response element was mapped between -168 and -151 base pairs 5' of the transcription start site. This element conforms with an inverted consensus glucocorticoid response element (GRE) but differs in two positions essential for protein DNA interaction between the GRE and the glucocorticoid receptor (GR). The induction of alpha 1 I3 gene promoter region constructs by dexamethasone was abolished by the receptor antagonist RU486, indicating that the GR participated in the activation of the alpha 1 I3 gene. In DNase I footprinting experiments with nuclear protein extracts from untreated and dexamethasone-treated FAZA cells, similar extents of alpha 1 I3 promoter upstream sequences were protected, indicating that proteins capable of binding in the glucocorticoid response-mediating element (GME) region were present before and after arrival of the hormonal signal. However, a purified recombinant fragment of the GR which contained essentially only its DNA binding domain was unable to bind at the GME although it interacted strongly with a consensus GRE sequence.

Acute-Phase Proteins↗
PubMed1991

The expression of interleukin-6 by a rat macrophage-derived cell line.

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rat macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.

Animals↗
PubMed1991

Gene regulation by interleukin 6.

Interleukin 6 (IL-6) is a central alarm hormone of the mammalian body. During acute and chronic inflammations, it induces acute phase plasma protein synthesis by liver hepatocytes, modulates the immune response and participates in the regulation of body temperature (fever). In addition, it is a growth factor for certain tumor cells, such as myeloma cells. The details of the IL-6 signal transduction mechanism are unknown. We have contributed to this problem at 2 levels: (a), we have mapped an IL-6-response element (IL-6-RE) in the 5' flanking region of the alpha 2-macroglobulin gene (alpha 2M), a prototype rat liver acute phase gene. This element, CTGGGA, serves as a binding site for nuclear factors that facilitate hormone induced transcription. We have begun to characterize these factors from hepatic cells and demonstrated that they undergo characteristic IL-6-induced changes. Similar factors were also discovered in human Burkitt tumor derived cell lines (B cells). These bound at the IL-6-RE of the rat alpha 1M gene and formed indistinguishable protein DNA complexes, as the corresponding hepatic factors. Thus, common elements probably operate in the IL-6 signal transduction cascade in liver cells and B cells; (b), we have cloned the rat liver IL-6 receptor (IL-6-R) and derived its amino acid sequence. It was 53% identical to the human leukocyte IL-6-R and all functional domains were highly conserved. Therefore, the cell-type specific responses to IL-6 in liver cells and lymphocytes were probably not due to cell-type specific forms of the receptor, but to other so far unknown elements of the signal transduction cascade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The Interleukin-6-dependent DNA-binding protein gene (transcription factor 5: TCF5) maps to human chromosome 20 and rat chromosome 3, the IL6 receptor locus (IL6R) to human chromosome 1 and rat chromosome 2, and the rat IL6 gene to rat chromosome 4.

Using two panels of somatic cell hybrids segregating either human or rat chromosomes, the gene encoding the interleukin-6-dependent DNA-binding protein, also called liver activator protein (designated transcription factor 5: TCF5), was assigned to human chromosome 20 and to rat chromosome 3. The TCF5 gene might be identical with the NF-IL6 gene. The locus encoding the IL6 receptor gene (IL6R) was localized to human chromosome 1 and rat chromosome 2. An IL6R-like (IL6RL) locus was also assigned to human chromosome 9. In addition, the rat interleukin-6 (IL6) gene was assigned to rat chromosome 4. These mapping data allow one to extend comparison between the rat, mouse, and human gene maps.

Animals↗
PubMed1991

Autocrine activity of interleukin 6 secreted by hepatocarcinoma cell lines.

Among several rat hepatoma cell lines known to secrete interleukin 6 (IL6), the HTC.JZ1 line stands out as a high-level producer. HTC.JZ1 cells were stimulated to secrete up to fourfold increased amounts of IL6 over 24 hours by treatment with lipopolysaccharides (LPS). Both functional IL6 levels, measured as hepatocyte stimulating factor (HSF) activity, and IL6 mRNA concentrations were increased proportionally by exposure to LPS. Similarly, IL6 mRNA was induced by LPS treatment in cultured primary rat hepatocytes. The induction of Il6 mRNA by LPS was inhibited both in primary hepatocyte and hepatoma cell cultures by treatment with the synthetic glucocorticoid dexamethasone, consistent with the known analogous repression of the IL6 gene by dexamethasone in macrophages, monocytes and fibroblasts. IL6 secreted by HTC.JZ1 cells was utilized as an autocrine inducer of endogenous acute phase gene expression: HTC cells expressed constitutive levels of alpha 2-macroglobulin (alpha 2M) mRNA specified by the major rat acute phase gene, the alpha 2M gene, which is known to be regulated by IL6. By contrast, normal rat liver biopsy material and a number of other rat hepatoma cell lines lacked endogenous IL6 production and showed very low to zero expression of endogenous alpha 2M mRNA. Expression of alpha 2M mRNA in HTC.JZ1 cells was inducible by treatment with LPS. The constitutive and the LPS-induced production of alpha 2M mRNA were significantly reduced (up to 50% inhibition) by addition of an anti IL6 serum to the culture medium and removal of the immune complexes. However, complete neutralization of the alpha 2M-inducing HSF activity could not be obtained with anti-IL6 serum alone, probably because HTC.JZ1 cells secrete comparable quantities of a second HSF activity. This activity, the cytokine leukemia inhibitory factor (LIF), is also known to stimulate transcription of the rat alpha 2M gene but was not reactive with anti-IL6 sera. The induction of IL6 mRNA in HTC cells by LPS was regulated at the transcriptional level, as demonstrated by a series of mutagenesis and transfection experiments. Progressive deletion of 5' flanking sequences from the IL6 gene promoter region reduced the basal level, and the LPS-induced promoter activity after transfection into HTC.JZ1 hepatoma cells. IL6 has been shown to act as an autocrine regulator of growth for certain B lymphoid cell lines derived from human multiple myelomas. The results presented here establish that IL6 secreted by certain hepatoma cell lines also acts in an autocrine fashion to induce expression of the endogenous acute phase alpha 2M gene.

Animals↗
PubMed1991

Interleukin 6 response factor binds co-operatively at two adjacent sites in the promoter upstream region of the rat alpha 2-macroglobulin gene.

Transcription of the alpha 2-macroglobulin gene (alpha 2M) in rat hepatocytes is strongly induced during acute inflammations by interleukin 6 (IL6). An IL6-response region has previously been mapped in the promoter upstream sequence of this gene. The region consists of two adjacent elements (IL6-REs), the IL6-RE core (CTGGGAA, -164 to -158 bp) and the core homology (CTGGAAA, -184 to -178 bp), elements, that are located 20 bp apart. Both elements bind nuclear factors with very similar protein-DNA contact patterns when they are contained in their original sequence context. A protein-DNA complex III was obtained in gel mobility shift experiments using a probe individually representing the core site. With probes containing both the core and core homology sites, a hormone inducible complex II of slower mobility was obtained. Complex II consisted of multiple copies of the same protein or proteins with very similar molecular masses bound at both sites. The core homology site was the weaker binding site. With a probe containing two tandem copies of the core site, binding at the second site occurred with 81 times greater affinity when the first site was occupied, than when it was free. Thus, the factor binding at the IL6-REs, the IL6-RE binding protein (IL6 RE-BP), was capable of co-operatively interacting with itself. Another factor, IL6-DBP/LAP, has recently been shown to be involved in the regulation of a major subgroup of acute phase genes by IL6. Using recombinant IL6-DBP/LAP and corresponding antisera, we demonstrated here that the IL6 RE-BP of the alpha 2M gene was distinct from IL6-DBP/LAP and from the related factor DBP. Thus, two major IL6-response elements can be distinguished: type 1 elements occurring in the human C-reactive protein, hemopexin and haptoglobin genes and utilizing IL6-DBP/LAP; and type 2 elements occurring in the rat alpha 2M, and alpha 1-acid glycoprotein genes, and utilizing a different IL6 RE-BP. The IL6 RE-BP of the alpha 2M gene was also shown to be distinct from the transcription factor NF kappa B. The IL6RE-BP had relative molecular mass of Mr = 46,000, distinct from IL6-DBP/LAP (Mr = 32,000) and NF kappa B (Mr = 50,000) and its overall DNA binding capacity was induced under acute phase conditions.

Animals↗
PubMed1991

Sequence of rat alpha 1-macroglobulin, a broad-range proteinase inhibitor from the alpha-macroglobulin-complement family.

The alpha-macroglobulin-complement family of plasma proteins includes at least three different alpha-macroglobulins in rats, alpha 2-macroglobulin (alpha 2M), alpha 1 inhibitor III (alpha 1I3), and alpha 1-macroglobulin (alpha 1M). These high molecular weight polypeptides (Mr 160,000 to 200,000) are broad-range proteinase inhibitors. They utilize an internal thiolester function to trap proteinases that cleave their bait regions. The amino acid sequences of alpha 2M and two variants of alpha 1I3 are known. The isolation of alpha 1M cDNA clones and the determination of their cDNA and derived amino acid sequences are reported here. Alpha 1M shares 57.2% and 53.0% overall amino acid sequence identity with alpha 2M and alpha 1I3, respectively, but differs significantly from both in its bait region, suggesting that each inhibitor addresses a different spectrum of proteinases. The disulfide bridge structure of alpha 1M was deduced from its sequence and showed extensive similarities with the experimentally determined structures of other alpha-macroglobulins, suggesting similar overall tertiary structures. Alpha 1M mRNA was detected in 13 different rat tissues tested, whereas alpha 2M and alpha 1I3 mRNAs showed a far more restricted tissue distribution. While alpha 2M and alpha 1I3 mRNA and protein concentrations are significantly altered during acute and chronic inflammations, alpha 1M plasma concentrations are changed only twofold. Thus, in contrast with alpha 2M and alpha 1I3, the functions provided by alpha 1M may be ubiquitously and constitutively required in a broad range of tissues.

Amino Acid Sequence↗
PubMed1990

Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor.

A series of cDNA clones coding for the rat liver interleukin 6 receptor (IL6-R) were isolated from an acute-phase library. The identity of the clones was established (a) by DNA sequence analysis and comparison with the known human leukocyte IL6-R and (b) by demonstrating that the clones generated specific IL6 ligand binding activity and IL6-dependent regulation of acute-phase gene control elements after transfection into appropriate recipient cells. Two types of cDNA clones were obtained corresponding to two mRNA species of different length, both encoding an identical protein of 462 amino acid residues. The two prototype clones, pRIL6RC.21 and pRIL6RC.6 contained 3'-untranslated regions of 550 and 3100 nucleotides, respectively. The sequence motifs TTATTTAT and ATTTA associated with the regulation of mRNA stability and translation efficiency were present only in the longer mRNA species. The deduced amino acid sequence of the rat liver IL6-R was 53% identical with the human leukocyte IL6-R. Both receptors contained conserved structural features in their extracellular domains, including the signal peptide, a C2 domain characteristic of the immunoglobulin superfamily, and two domains shared among members of a family of cytokine and growth factor receptors. The strongly conserved intracellular portion of the rat liver IL6-R lacked recognizable signal transduction domains. The cDNA clones were used to demonstrate that rat liver IL6-R mRNA concentrations were increased 4.2-fold at 12 h after the induction of an experimental acute-phase response. Clone pRIL6R.21ex, but not clone pRIL6RC.6ex, generated specific IL6 ligand binding activity after transfection into human Jurkat cells that lack the endogenous IL6-R. By contrast, only pRIL6RC.6ex reconstituted a response of human Hep3B-2 and HepG2 hepatoma cells to mouse IL6. These human hepatoma cells were highly responsive to human IL6 but did not respond to physiologic concentrations of murine IL6. After cotransfection with pRIL6RC.6ex and plasmids containing the chloramphenicol acetyltransferase reporter gene under the control of IL6 response elements of acute-phase plasma protein genes, these cells showed a strong stimulation of the reporter gene by recombinant mouse IL6. Thus, both cell surface ligand binding activity and the complete IL6 signal cascade terminating in the transcriptional induction of IL6-dependent promoters were successfully reconstituted. Therefore, both IL6-R mRNA species code for functionally active receptor, depending on the target cell, but only the longer mRNA species coded for significant receptor levels in human hepatoma cell lines.

Amino Acid Sequence↗
PubMed1990

Alpha-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system.

Significant primary sequence homology between the alpha-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus alpha-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus alpha-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus alpha-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general "trapping" mechanism shared by most alpha-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus alpha-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus alpha-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the alpha-macroglobulin family. Because both properties are contained in one molecule, we propose the name "limac" to describe this Limulus alpha-macroglobulin complement-like protein.

Amino Acid Sequence↗
PubMed1990

IL-6 functions as an exocrine hormone in inflammation. Hepatocytes undergoing acute phase responses require exogenous IL-6.

We have previously shown that IL-6 is the major monocyte- and fibroblast-derived regulator of acute phase protein gene expression and synthesis in hepatocytes in inflammation. Recently, we and others have shown that rat and human hepatoma cells express IL-6 mRNA, and the question arose as to whether normal hepatocytes express IL-6 and whether any such expression occurs under normal physiologic conditions or is seen in inflammation. Poly A+ mRNA of liver from normal rats and from rats undergoing an acute phase response was not positive when probed with cDNA for rat IL-6 under conditions in which macrophage mRNA was strongly positive. We then compared poly A+ mRNA from purified hepatocytes freshly isolated from normal rats--from rats that were undergoing an acute inflammatory response and from freshly isolated normal hepatocytes that had been cultured for 24 h in the presence or absence of dexamethasone (microM). Only the mRNA from normal hepatocytes cultured for 24 h in the absence of any glucocorticoid was obviously positive for IL-6. The increased expression of gamma-fibrinogen mRNA indicated the presence of inflammation. These results confirm the identification of IL-6 as an exogenous hormone for regulating normal hepatic acute phase protein synthesis in inflammation and rules out an autocrine mechanism being active in the liver in normal homeostasis.

Acute-Phase Reaction↗
PubMed1990

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

Interleukin 6 (IL-6) was established as a transcriptional inducer of the rat alpha 2-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 defined 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 acute-phase 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.

Animals↗
PubMed1990

The assignment of the genes coding for human complement components C6 and C7 to chromosome 5.

A panel of 19 somatic cell hybrids was tested for the presence of human sequences coding for complement components C6 and C7 by restriction enzyme digestion and Southern blots probed with human C6 and C7 cDNA probes. C7 was also detected by amplifying part of the human gene in hybrid DNA using the polymerase chain reaction. Detection of human C6 and C7 was completely correlated with the presence of chromosome 5.

Animals↗
PubMed1990

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

We have cloned the promoter for the human third component of complement (C3) gene and have identified sequences involved in its regulation during the acute-phase response. A construct linking 199 bp of the C3 promoter to the firefly luciferase gene was found to be very responsive to interleukin-1 (IL-1) and modestly responsive to interleukin-6 (IL-6) by transfection analysis in the human hepatoma line Hep3B2. Simultaneous treatment with the two cytokines showed a strong synergy between the actions of the two molecules. A 58-bp fragment (-127 to -70 bp) was shown by 5' and 3' deletional mutagenesis to contain cis-acting elements that mediated both the IL-1 response and the IL-1-plus-IL-6 synergistic response of this promoter. When coupled to a heterologous promoter, this fragment enabled the synergistic induction by IL-1 plus IL-6. Sequences homologous to the palindrome ACATTGCACAATCT, which mediates the induction of the IL-6 gene by IL-1 (S. Akira, H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto, EMBO J. 9:1897-1906, 1990), and the core sequence of the IL-6-responsive element of the rat alpha 2-macroglobulin gene (CTGGGA; M. Hattori, L. J. Abraham, W. Northemann, and G. H. Fey, Proc. Natl. Acad. Sci. USA 87:2364-2368, 1990) are contained within this fragment in immediate juxtaposition and partially overlapping. Site-directed mutagenesis within this homology region drastically reduced the inducibility of the C3 promoter by either cytokine. DNase I footprinting analysis defined a binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP), which included the IL-1-responsive element-like sequence. No differences were seen between the footprints generated by using extracts from unstimulated and IL-1-stimulated Hep3B2 cells. However, gel retardation analyses revealed two IL-1-specific bands. The data suggest that the induction by IL-1 is mediated by a factor belonging to the family of C/EBP-related proteins.

Animals↗