Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes

The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.     

Characterization of soluble gp130 released by melanoma cell lines: A polyvalent antagonist of cytokines from the interleukin 6 family

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.     

Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption

Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity.     

Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb

The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6-dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.     

Wool production in transgenic sheep: results from first-generation adults and second-generation lambs

The neuropoietic cytokines of the interleukin-6 family are a group of structurally and functionally related polypeptides. We studied the effect of the multifunctional neuropoietic cytokines, including oncostatin M (OSM), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), on anaplastic glioma cell lines. Growth and morphology of the glioma cell lines were affected differently. While IL-6 and LIF exerted no or only small minor morphological changes and growth retardation, OSM induced a marked change in morphology and a strong suppression of growth. OSM treated cells were characterized by enlargement and the formation of multiple, thin processes thus resembling mature cultured astrocytes. The growth inhibitory effects were dose dependent with a maximum exerted by addition of 50 ng/ml OSM. The inhibition of DNA synthesis by OSM could be abolished by antibodies blocking either the activity of OSM or the OSM-receptor component, gp130.     

An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M

The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM). Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signal-transducing subunit shared with interleukin-6 and interleukin-11. The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF. In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands. On cells that express LIF-R and gp130, all LIF-R ligands were antagonized. On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R. Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist. The antagonist is therefore likely to work by preventing receptor oligomerization.     

IFN-alpha is a survival factor for human myeloma cells and reduces dexamethasone-induced apoptosis

IFN-alpha is used as a maintenance therapy in patients with multiple myeloma, but its benefit is a matter of controversy. In vitro studies show that IFN-alpha can both stimulate and inhibit myeloma cell proliferation. We have tested the effect of IFN-alpha on the survival of myeloma cell lines and primary plasma cells. IFN-alpha significantly reduced the apoptosis induced by removal of IL-6 in four IL-6-dependent myeloma cell lines. It also reduced the level of apoptosis induced by dexamethasone in these cell lines as well as in purified primary myeloma cells from seven patients. IFN-alpha promoted the survival of myeloma cells, which, following removal of IL-6, were blocked in G1 and died. However, unlike IL-6, IFN-alpha-treated cells remained mainly blocked in the G1 phase of the cycle. While the effects of IL-6 are mediated through stimulation of its gp130 receptor subunit, the IFN-alpha-induced survival of myeloma cells was independent of gp130 transducer activation (as demonstrated using a neutralizing anti-gp130 Ab). However, the signal transduction cascades activated by these two cytokines share at least some common elements, since stimulation with either IFN-alpha or IL-6 resulted in STAT3 phosphorylation. These results indicate that IFN-alpha promotes the survival, but not the proliferation, of myeloma cells, preventing the apoptosis induced by removal of IL-6 or addition of dexamethasone. This survival factor activity may explain the conflicting reports on the effects of IFN-alpha on myeloma cell proliferation.     

Osteoclasts are present in gp130-deficient mice

Interleukin (IL)-6, IL-11, leukemia inhibitory factor, and oncostatin M similarly induce osteoclast formation in cocultures of osteoblastic cells and bone marrow cells. These cytokines share a common signal transducer, gp130, which forms a receptor complex with the specific receptor for each cytokine. To investigate the role of gp130 in osteoclast development, we examined bone tissues in gp130-deficient and wild-type newborn mice of the ICR background. Soft x-ray radiographs and microfocus x-ray computed tomographs revealed that bone marrow cavities were present in tibiae and radii of both wild-type and gp130-deficient mice. Microfocus x-ray computed tomography and histological examination demonstrated a decrease in the amount of trabeculae at the metaphysial region in tibiae and radii of the gp130-deficient mice compared with the wild-type mice. The number ofosteoclasts in gp130-deficient mice was about double that in the wild-type mice. There were no apparent differences in the distributions of alkaline phosphatase-positive osteoblasts and the osteoid surface on the trabecular bone at the metaphysial region between the wild-type and gp130-deficient mice. The volume of mineralized trabecular bones was also decreased at mandibulae, accompanied by the increased number of osteoclasts in gp130-deficient mice compared with the wild-type and heterozygous mice. These results suggest that the formation of osteoclasts is not solely dependent on gp130 signaling, at least during fetal development. The osteoclastic bone resorption in gp130-deficient mice may be caused by the functional redundancy of bone-resorbing hormones and cytokines other than those of the IL-6 family.     

Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation

Interleukin-6 (IL-6) induces growth arrest and macrophage differentiation through its receptor in a murine myeloid leukaemic cell line, M1, although it is largely unknown how the IL-6 receptor generates these signals. By using chimeric receptors consisting of the extracellular domain of growth hormone receptor and the transmembrane and cytoplasmic domain of gp130 with progressive C-terminal truncations, we showed that the membrane-proximal 133, but not 108, amino acids of gp130 could generate the signals for growth arrest, macrophage differentiation, down-regulation of c-myc and c-myb, induction of junB and IRF1 and Stat3 activation. Mutational analysis of this region showed that the tyrosine residue with the YXXQ motif was critical not only for Stat3 activation but also for growth arrest and differentiation, accompanied by down-regulation of c-myc and c-myb and immediate early induction of junB and IRF1. The tight correlation between Stat3 activation and other IL-6 functions was further observed in the context of the full-length cytoplasmic region of gp130. The result suggest that Stat3 plays an essential role in the signals for growth arrest and differentiation.     

Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects

The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP-mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.     

Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: a combined in vitro and in vivo analysis

By means of a combined in vitro and in vivo analysis we provide evidence that IL-1 beta and PDGF-B, but not OSM (oncostatin M) or IL-6, are major mitogens for the spindle cells of Kaposi's sarcoma (KS) in vivo. PDGF-B and IL-1 beta stimulated proliferation of cultivated KS spindle cells in vitro. Analysis of gene expression in vivo revealed that both factors as well as the PDGF beta-receptor are present in KS lesions. By contrast, IL-6 had no effect and OSM inhibited proliferation of cultivated KS spindle cells. Again, the effect of these factors on cultivated KS spindle cells in vitro was reflected by the gene expression observed in KS lesions in vivo. Neither the expression of IL-6 receptor nor of OSM could be detected in KS lesions by in situ hybridization. Moreover, in situ hybridization revealed an identical pattern of gene expression in cultivated KS spindle cells and KS spindle cells in vivo with respect to the above-mentioned cytokines [PDGF-B, IL-1 beta, IL-1 alpha, IL-6, OSM] and their receptors [PDGF beta-receptor, gp130, IL-6 receptor, leukemia inhibitory factor (LIF) receptor]. This further supported the suitability of cultivated KS spindle cells as an in vitro model in order to determine which cytokines may activate proliferation of KS spindle cells in vivo.