Interleukin-11 signals through the formation of a hexameric receptor complex

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.     

The study of gp130/the inflammatory factors regulating osteoclast differentiation in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic immune disease characterized by synovitis and bone destruction. The osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis. In this paper, we report that Interleukin (IL)-6, IL-6Rα/gp130, IL-11, IL-27, and Matrix Metallo Proteinases (MMP)-9 expression results in serum of the RA group were significantly higher than that of the control group. The gp130 positive cells in peripheral blood mononuclear cell (PBMC) and osteoclast-like cells (OLC) which had been induced with receptor activator of nuclear factor κB ligand (RANKL) in RA group were also higher than that in the control group. In addition, after OLC in RA group is cultured with anti-gp130 Monoclonal antibody (McAb), the IL-6 and MMP-9 expression in osteoclast supernatant insignificantly decreased. Meanwhile, the expression results of Tartrate Resistant Acid Phosphatase (TRAP)-positive cells and osteoclasts were also decreased significantly. Our study suggests that regulating gp130 receptor can be used to control the differentiation and formation of osteoclasts, which provides a new clinical strategy for RA patients in the future.     

The soluble Interleukin 6 receptor: generation and role in inflammation and cancer

Soluble cytokine receptors are frequently found in human serum, most of them possessing antagonistic properties. The Interleukin 6 receptor (IL-6R) is found as a transmembrane protein on hepatocytes and subsets of leukocytes, but soluble isoforms of the IL-6R (sIL-6R) are generated by alternative splicing or by limited proteolysis of the A Disintegrin And Metalloproteinases (ADAM) gene family members ADAM10 and ADAM17. Importantly, the sIL-6R in complex with its ligand Interleukin 6 (IL-6) has agonistic functions and requires cells expressing the signal transducing ß-receptor gp130 but not the membrane-bound IL-6R. We have called this process IL-6 trans-signaling. Naturally occurring isoforms of soluble gp130 (sgp130), which are generated by alternative splicing, are natural inhibitors of IL-6 trans-signaling, leaving IL-6 classic signaling via the membrane-bound IL-6R unaffected. We used recombinant sgp130Fc protein and recently generated transgenic mice expressing high levels of sgp130Fc to discriminate between classic and trans-signaling in vivo, and demonstrated that IL-6 trans-signaling is critically involved in generation and maintenance of several inflammatory and autoimmune diseases including chronic inflammatory bowel disease, rheumatoid arthritis, peritonitis and asthma, as well as inflammation-induced colon cancer.     

Identification of three distinct receptor binding sites of murine interleukin-11

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.     

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.     

Detection of direct binding of human herpesvirus 8-encoded interleukin-6 (vIL-6) to both gp130 and IL-6 receptor (IL-6R) and identification of amino acid residues of vIL-6 important for IL-6R-dependent and -independent signaling

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.     

The Amino Acid Exchange R28E in Ciliary Neurotrophic Factor (CNTF) Abrogates Interleukin-6 Receptor-dependent but Retains CNTF Receptor-dependent Signaling via Glycoprotein 130 (gp130)/Leukemia Inhibitory Factor Receptor (LIFR)

Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR·gp130·LIFR or IL-6R·gp130·LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. Arg²⁸ is in close proximity to the CNTFR binding site. Using molecular modeling, we hypothesized that Arg²⁸ might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR-binding site from cardiotrophin-like cytokine to CNTF. Cardiotrophin-like cytokine selectively signals via the CNTFR·gp130·LIFR complex, albeit with a much lower affinity compared with CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2, and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR·gp130·LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R·gp130·LIFR. Quantification of CNTF-dependent proliferation of CNTFR·gp130·LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.     

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1-D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1-D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein - consisting of two extracellular gp130 regions (D1-D6) dimerised by an IgG1-Fc part - to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1-D3 with respect to the membrane-proximal domains D4-D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.     

Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses.

Signal transduction in response to interleukin-6 (IL-6) requires binding of the cytokine to its receptor (IL-6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL-6 and soluble IL-6R (sIL-6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL-6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL-6 and sIL-6R. We created recombinant sgp130 proteins that showed binding to IL-6 in complex with sIL-6R and inhibited IL-6/sIL-6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL-6 stimulated proliferation of BAF/3 cells expressing gp130 and membrane bound IL-6R, indicating that sgp130 did not interfere with IL-6 bound to IL-6R on the cell surface. Additionally, sgp130 partially inhibited proliferation induced by leukemia inhibitory factor (LIF) and oncostatin M (OSM) albeit at higher concentrations. Recombinant sgp130 protein could be used to block the anti-apoptotic effect of sIL-6R on lamina propria cells from Crohn disease patients. We conclude that sgp130 is the natural inhibitor of IL-6 responses dependent on sIL-6R. Furthermore, recombinant sgp130 is expected to be a valuable therapeutic tool to specifically block disease states in which sIL-6R transsignaling responses exist, e.g. in morbus Crohn disease.     

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.     

Identification of amino acid residues of gp130 signal transducer and gp80 alpha receptor subunit that are involved in ligand binding and signaling by human herpesvirus 8-encoded interleukin-6

Human herpesvirus 8-encoded interleukin-6 (vIL-6) signals through the gp130 signal transducer but is not dependent on the IL-6 receptor alpha subunit (IL-6R, gp80) that is required for signaling by endogenous IL-6 proteins; however, IL-6R can enhance vIL-6 activity and can enable signaling through a gp130 variant, gp130.PM5, that is itself unable to support vIL-6 signaling. These findings suggest that the vIL-6-gp130 interactions are qualitatively different from those of human IL-6 (hIL-6) and that vIL-6 signaling may be more promiscuous than that of hIL-6 but that IL-6R may play a role in vIL-6 signaling in vivo. To examine the receptor binding requirements of vIL-6, we have undertaken mutational analyses of regions of gp130 and IL-6R potentially involved in interactions with ligand or in functional complex formation and used these variants in functional, ligand-binding, and receptor dimerization assays. The data presented identify positions within two interstrand loops of the gp130 cytokine-receptor homology domain that are important for vIL-6 signaling and vIL-6-induced receptor dimerization and show that vIL-6, like hIL-6, can form complexes with IL-6R and gp130 but that the roles of putative cytokine-binding residues of IL-6R in ligand-induced functional complex formation are qualitatively different in the case of vIL-6 and hIL-6.     

Structure-guided optimization of the interleukin-6 trans-signaling antagonist sgp130

Binding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation.     

STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone.

Interleukin (IL)-6-type cytokines stimulate osteoclastogenesis by activating gp130 in stromal/osteoblastic cells and may mediate some of the osteoclastogenic effects of other cytokines and hormones. To determine whether STAT3 is a downstream effector of gp130 in the osteoclast support function of stromal/osteoblastic cells and whether the gp130/STAT3 pathway is utilized by other osteoclastogenic agents, we conditionally expressed dominant negative (dn)-STAT3 or dn-gp130 in a stromal/osteoblastic cell line (UAMS-32) that supports osteoclast formation. Expression of either dominant negative protein abolished osteoclast formation stimulated by IL-6 + soluble IL-6 receptor, oncostatin M, or IL-1 but not by parathyroid hormone or 1,25-dihydroxyvitamin D3. Because previous studies suggested that IL-6-type cytokines may stimulate osteoclastogenesis by inducing expression of the tumor necrosis factor-related protein, receptor activator of NF-kappaB ligand (RANKL), we conditionally expressed RANKL in UAMS-32 cells and found that this was sufficient to stimulate osteoclastogenesis. Moreover, dn-STAT3 blocked the ability of either IL-6 + soluble IL-6 receptor or oncostatin M to induce RANKL. These results establish that STAT3 is essential for gp130-mediated osteoclast formation and that the target of STAT3 during this process is induction of RANKL. In addition, this study demonstrates that activation of the gp130-STAT3 pathway in stromal/osteoblastic cells mediates the osteoclastogenic effects of IL-1, but not parathyroid hormone or 1, 25-dihydroxyvitamin D3.     

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.