Gene regulation by interleukin 6

Bio~'himie ( 1991 ) 73, 47-50 O Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris

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Gene regulation by interleukin 6 GH Feyl, M Hattori*, G Hockel, T Brechner , G Baffet , M Baumann**, H Baumann2, W Northemann*** /Department t~ lmmunology. Research Institute of Scripps Clinic, •0666 North Torrev Pines Rd. La Jolla. CA 92037: 2Department of Molecular and Celhdar Biology. Roswell Park Cancer Institute. Buffalo, NY 14263, USA (Received 3 October 1990; accepted 23 November 1990)

Summary - - 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 ot2-macroglobulin gene (ot2M), 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 ot.tM 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 ceil-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. Two mRNA species (cDNA clones) for the rat liver IL-6-R were isolated. Both coded for an identical protein and differed in their 3' untranslated regions (3' UTR), due to alternative polyadenylation. The 3' UTR of the longer mRNA species carried sequence elements associated with regulation of mRNA stability and translation efficiency. Upon transfection into hepatic cells and Jurkat cells (leukocytes), the shorter mRNA species selectively functioned in the Jurkat cells and only the longer mRNA species in hepatic cells. IL-6-R mRNA levels were upregulated 4-fold during an acute phase response. The receptor mRNA was inducible by glucocorticoids and under negative regulation by IL-6. IL-6-R mRNA was further inducible in HL60 promyelocytic cells upon induction of granulocytic differentiation. interleukin 6 / cytokines / receptors / gene regulation / inflammation

Interleukin 6 (IL-6) is a central alarm hormone of the h u m a n body. Its multiple roles in the host defense to tissue injury and infection have recently been reviewed [1-9]. A m o n g its m a n y functions in coordinating diverse facets of host defense, its role in inducing the synthesis of protective plasma proteins (the acute phase proteins) by the liver is one of the best studied [1, 4, 6-9]. IL-6 also modulates the i m m u n e response and is involved in

*Present address: Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan **Present address: Boehringer Mannheim AG, Mannheim, Germany ***Present address: Elias, GMBH, Freiburg, Germany Abbreviations: IL-I and IL-6, interleukins 1 and 6; ct2M, ot2macroglobulin; IL-6-R, interleukin 6 receptor, ligand binding chain; IL-6-RE, interleukin 6-response element; gpl30, glycoprotein 130 (the 130 kDa signal transduction chain of the IL-6-R); LIF, leukemia inhibitory factor

the terminal differentiation of B lymphocytes to plasma cells and the accompanying induction of immunogiobuhn synthesis [10], as well as the maturation of thymocytes [1-3]. It has recently been recognized as an important regulator of body temperature (fever) [1 1], and the roles of IL-6 in the central nervous system constitute an area of intense current research. IL-6 has recently been shown to be expressed in murine blastocysts prior to the onset of hemopoiesis, and thus it is reasonable to expect that IL-6 may also play an as yet undetermined role in early embryogenesis [12]. Finally, IL-6 is secreted by m a n y tumor-derived ceil-lines and in particular by lines derived from human multiple myelomas. This endogenous hormone is reinternalized via the IL-6 receptor and utilized by some of these cell lines as an essential autocrine growth factor [13]. It has therefore been proposed that IL-6 may play a role as an autocrine growth factor in the pathogenesis of certain human tumors, in particular multiple m y e l o m a s [ ! 3].

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In spite of the many important roles of this hormone, verx little is knoxvn about the signal transduction cascade from the cell-surface membrane to specific target genes in the nucleus. The IL-6 signal is first received by a cell surface receptor protein 114-161 as is the case for most other known peptide hormones. However, unlike most other peptide hormone receptors, this receptor does not seem to propagate the signal into one of the 2 major well-known categories of second messenger pathways, ie the pathway used by G-protein associated receptors and the pathway triggered by receptors with tyrosine protein kinase activities. Thus, IL-6 activates at, apparently novel type of pathway. A cDNA coding lbr the human lymphocyte IL-6 receptor ligand binding chain has recently been cloned and the amino acid sequence of the receptor was deduced [15[. No sequence homology with the G-protein association and the protein tyrosine kinase domains of other receptors was found, further supporting the hypothesis that the IL-6 signal utilizes a novel class of receptor and signal transduction pathways. The ligand binding chain of the IL-6 receptor (IL-6-R), requires the association of a second protein, gpl30, for successful signal transmission [16]. The IL-6-R itself has a transmembrane domain and a short cytoplasmic domain, but the cytoplasmic domain does not transduce the signal and is dispensable [16]. A truncated 'soluble" mutant of the IL-6-R, lacking the transmembrane and cytoplasmic domains, was still found to function [ 16]. The IL-6 hormone triggers the association between the IL-6-R and the gpl30 transducer protein. This protein also has a transmembrane domain and a longer cytoplasmic domain with recognizable homology to a G-protein association domain, as is found in p21 ras proteins [171. Thus. the hormonal signal is generated by the hormone induced association ol tt,-o-K and gpi30, and is propagated into the cytoplasm through the cytoplasmic domain of the gpl30 signal transducer. This represents a novel category of receptors and signal pathways, because it is one of the first known cases in which the ligandbinding domain and the signal transduction domains are carried on 2 separate polypeptide chains. Interestingly, both the IL-6-R and the gpl30 protein carry conserved domains with a number of other cytokine receptors [17, 18], suggesting that they have either evolved from common ancestors by divergent evolution, or possibly that exon shuffling has played a role in their evolution. One of the striking features of IL-6 is that the same hormone causes so many different cell-type specific responses in different types of target cells. The origin of this cell-type specificity is unknown. Possible explanations include: a), cell-type specific surface receptors (iigand-binding chains, signal transduction chains); b), cell-type specific cytoplasmic; and c), cell-type specific nuclear response elements (transcription factors, etc) of the signal cascade. Taga et a! had observed that

Fey ~'t a/ the high affinity IL-6 receptors on many different cell types had very similar ligand affinities. These authors therefore ?roposed that only one universal ligand binding chain may be present in different target cells, and that the diversity arises at a post-receptor level [14]. To test this hypothesis, we have cloned and sequenced the rat liver IL-6-R cDNA and deduced the protein sequence [18]. The sequence comparison suggests that the IL-6-R is highly conserved between human lymphocytes and rat hepatocytes. Moreover, the rat IL-6-R gene was mapped in collaboration with P and J Szpirer et al and only one gene was found [19]. These results suggested that the IL-6-R ligand binding chain is probably universal, and that some other post-receptor step in the signal transduction cascade is likely to be responsible for the cell-type specificity of the response to IL-6. Results Characterization t~'the rat hepatocyte IL-6 receptor

A cDNA library prepared from differentiated HL60 human promyelocytic cells that express high levels of the IL-6-R [14] was screened with a 60 bp synthetic oligonucleotide, derived from the human lymphocyte IL6-R sequence [151. We isolated a clone, pHIL6R.3, with a 3095 bp insert and sequenced it. The sequence was identical to the previously published sequence [15]. To generate a probe that contained non-repetitive coding sequences only, a 1209 bp Sphl fragment comprising only protein coding sequences was subcloned into the bluescript KS vector, generating plasmid pHIL-6R.32. This latter clone was used to screen rat liver acute phase eDNA libraries, and a series of clones whh overlapping inserts were isolated. The total cDNA sequence was derived, and shown to be 71% identical on the nucleotide level in the protein coding region with the human lymphocyte IL-6-R [I 5], and 53% identical on the amino acid level. The definitive identity of these clones as IL-6R clones was established by additional experiments, described below, showing that these clones coded for a functionally active receptor, generating specific ligand binding activity. The sequence homology between the human leukocyte and rat liver IL-6-R proteins extended along the entire length of the 2 proteins (462 amino-acid residues, including the signal peptide) and was particularly strong hi the signal peptide and transmembrane domains, but also in the extracellular portion and even in the cytoplasmic domain, although we know of no selectable function for this latter domain. The extracellular portion contains a so-called C2-domain, a conserved domain shared with other members of the immunoglobulin superfamily [20], and 2 domains, A and B, shared with a new superfamily of cytokine receptors [21]. All these data taken together suggest that the rat liver and

Gone regulation by intcrlcukin t~ human leukocyte IL-6-Rs are as closely conserved in their sequences as can be expected across the species boundaries. There is thus no evidence for the existence of cell-type specific IL-6-Rs. cDNA clones corresponding to 2 differem size classes of IL-6-R mRNA were isolated and sequenced. Both cDNAs contained poly-A tails and the complete protein coding sequences, coding for identical polypeptides. Both contained a short 5' untranslated region, but differed in the lengths of their 3' untranslated regions. Clone pRIL6-RC.6 had a long 3' untranslated region, close to 3 000 nucleotides. By contrast, clone pRIL-6RC.21 had a short 3' untranslated region of approximately 500 nucleotides. This latter clone was generated from the same gene by alternative polyadenylation at a minor polyadenylation signal [18]. By transfection studies into cultured cells it was demonstrated that both clones coded for functionally active receptor, but that they were utilized differentially in cultured lymphoid and hepatic cell lines. Both inserts were subcloned into mammalian expression vectors and transfected either into the T-cell line Jurkat or the hepatoma cell line HepG2. Specific ligand-binding activity was measured in Jurkat cells by exposing the transfected cells in a transient expression assay to radioiodinated IL-6. Specific ligand binding activity in Jurkat cells was generated only by transfection with the short clone pRIL6-RC.21, but not after transfection with the long clone. By contrast, after transfection into human HepG2 hepatoma cells, only the long but not the short clone generated functional activity. This is consistent with the observation that in rat livers predominantly the longer 4.5 kb IL-6-R mRNA species is detected. In acute phase rat livers a heterogeneous IL-6-R mRNA population is present, but the 4.5-kb long mRNA species is by far the most abundant one. Although we demonstrated a ceiltype specific utilization of the short and long IL-6-R mRNA species in cultured cell lines, the in vivo significance of this finding remains to be determined. The levels of IL-6-R mRNA can be regulated by external signals in hepatic cells. A 3 to 4-fold increase in receptor mRNA levels was observed in acute phase rat livers at various times after inducing the experimental inflammation, and peak levels were seen at 12-18 h, at the peak of the acute phase reaction. This induction is probably mediated by glucocorticoids, since we and others have observed in experiments with cultured hepatic cells that IL-6-R mRNA levels can be regulated by glucocorticoids [22, 231. We further observed that IL6-R mRNA levels can be induced by external signals in the human promyelocytic cell line HL60 after induction of granulocytic differentiation [ 181.

Nuclear endpoint of the IL-6 signal cascade We and others have mapped II-6-response elements (IL-6-REs) in the promoter upstream regions of major

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liver acute phase genes [4, 8, 9, 241. Our group has focussed on the rat o¢,-macroglobulin (ot_~M) gene. the prototype rat liver acute phase gene. By mutagenesis and transfection into hepatoma cells, we have confined the main IL-6-RE of the o~,M gene to an 18-bp sequence and shown that this element serves as a binding site for nuclear proteins. These proteins pre-exist in the hepatocyte nuclei prior to the arrival of the hormonal signal, and are altered by the hormone in a way that is recognizable by an altered mobility of the specific protein-DNA complex in so-called gel mobility shift experiments [24]. Recently, we have also detected the same proteins in human hepatoma cells, and protein-DNA complexes of indistinguishable mobility are induced by IL-6 in both the rat and human cells. We have demonstrated that the IL-6-RE of the rat 0¢zM gene not only binds the corresponding proteins from human hepatic cells, but that this sequence is also active in mediating the IL-6 response in human cells. To this effect constructs were prepared that carried 4 tandem copies of the 18-bp sequence in the same orientation in a plasmid, driving an SV40 early enhancerless promoter and a luciferase reporter gene. These constructs were transfected into human hepatoma cells, and treated with IL-6. As a result, this 18-bp sequence was sufficient to confer functional IL-6 responsiveness in human cells to a heterologous promoter in cis. This IL-6-RE sequence is different from those reported for the major human acute phase genes by other authors, and the protein binding there is different. The protein binding at this IL-6-RE is different from both NF~cB and the liver activation protein (LAP), although the binding site resembles a consensus NFlcB site. In living rats and most cultured hepatic cell systems the full induction of liver acute phase genes requires both IL-6 and glucocorticoids, although we have shown •

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to induce transcription from the rat o~,M promoter [24]. Recently we have found that the same 18-bp IL-6-RE sequence described above mediates in human hepatoma cells not only the IL-6-effect, but also the synergistic effect of IL-6 and giucocorticoids. This is surprising, because the 18 bp sequence does not contain consensus binding sites for the glucocorticoid receptor. The result suggests that either the glucocorticoid effect is indirect. or that the glucocorticoid receptor acts through this 18-bp sequence in a novel way. This could be for example a DNA interaction at a sequence that differs from the consensus glucocorticoid receptor binding site (GRE), or binding of the receptor not to DNA directly, but to the IL-6 responsive transcription factors bound at the IL-6-RE. Both these intriguing possibilities are currently being tested. Recently we reported on the production and secretion of IL-6 by a number of rat heoatoma cell lines [25]. The c~ tokine leukemia inhibitory factor (LIF) has a different structure than IL-6, but it induces an almost identical spectrum of acute phase genes in rat liver [26], and

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GH Fey et al

shares man,, common properties with IL-6. We have no,,~ found [hat some of the same rat hepatoma cell lines that secrete IL-6 also secrete LIF, and that both IL-6 and LIF are inducible in these cell lines by very similar treatments. This result raises the intriguing question of whether not only IL-6, but also LIF could possibly be involved in au,'ocfine regulatory mechanisms of hepatoma cells. We are currently investigating whether IL-6 or L1F or both, that are secreted by these hepatoma cells, play a role as autocrine growth factors or differentiation inducing factors in these hepatoma cells. If this were the case, then a possible role of these autocrine hormones in the pathogenesis of hepatocarcinoma would have to be investigated. In conclusion, the regulation of the IL-6 gene and the IL-6 receptor needs to be studied because both are involved in important processes in inflammation, development and possibly carcinogenesis. Manipulation of this receptor may permit the design of novel substances of pharmacological value, which may possibly be of use as anti-inflammatory drugs.

Acknowledgments We thank R Fletcher, D Barry and A Goei for expert technical assistance and K Dunn for the preparation of the manuscript.This study was supported by Public Health Service grants Ai22166 and A123351 (to GHF), A127493 (to WN) and CA261122 (to HB). MB. GH and TB were recipients of postdoctoral fellowships from the DFG (Deutsche Forschungsgemeinschaft), DAAD (Deutscher Akademischer Austauschdienst) and NATO, respectively. GB was partially supported by a special fellowship from the American Liver Foundation, and from the Philippe Foundation; and the CNRS, the French governmental research organization. This is a publication No 6573-IMM from the Department of Immunology, Research Institute of Scripps Clinic.

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Williams AF, Barclay AN (1988) The immunoglobulin superfamily-domains for cell surface recognition. Ann Rev lmmunol 6, 381--405 Bazan F (1988) Structural design and molecular evolution of a cytokine receptor superfamily. Proc Nat! Acad Sci USA 87, 6934-6938 Bauer J, Lengyel G, Bauer TM, Acs G, Gerok W (1989) Regulation of interleukin 6 receptor expression in human monocytes and hepatocytes. FEBS Lett 249, 27-30 Snyers L, DeWit L, Content J (1990) Giucocorticoid upregulation of high affinity interleukin 6 receptors on human epithelial cells. Proc Nail Acad Sci USA 87, 2838-2842 Hattori M, Abraham LJ; Northemann W, Fey GH (1990) Acute phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2 macroglobulin gene. Proc Nail Acad Sci USA 87, 2364-2368 Northemann W, Hattori M, Baffet G, Braciak TA, Fletcher RG, Abraham LJ, Gauldie J, Baumann M, Fey GIh (1990) Production of interleukin 6 by hepatoma cells. Mol Biol Med 7, 273-285 Baumann H, Wong GG (1989) Hepatocyte-stimulating factor III shares structural and functional identity with leukemia inhibitory factor. J lmmunol 143, 1163-1167