Receptor subunit-specific action of oncostatin M in hepatic cells and its modulation by leukemia inhibitory factor

The related cytokines, interleukin-6 (IL-6), oncostatin M (OSM), and leukemia inhibitory factor (LIF) direct the formation of specific heteromeric receptor complexes to achieve signaling. Each complex includes the common signal-transducing subunit gp130. OSM and LIF also recruit the signaling competent, but structurally distinct OSMRbeta and LIFRalpha subunits, respectively. To test the hypothesis that the particularly prominent cell regulation by OSM is due to signals contributed by OSMRbeta, we introduced stable expression of human or mouse OSMRbeta in rat hepatoma cells which have endogenous receptors for IL-6 and LIF, but not OSM. Both mouse and human OSM engaged gp130 with their respective OSMRbeta subunits, but only human OSM also acted through LIFR. Signaling by OSMRbeta-containing receptors was characterized by highest activation of STAT5 and ERK, recruitment of the insulin receptor substrate and Jun-N-terminal kinase pathways, and induction of a characteristic pattern of acute phase proteins. Since LIF together with LIFRalpha appear to form a more stable complex with gp130 than OSM with gp130 and OSMRbeta, co-activation of LIFR and OSMR resulted in a predominant LIF-like response. These results suggest that signaling by IL-6 cytokines is not identical, and that a hierarchical order of cytokine receptor action exists in which LIFR ranks as dominant member.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

Characterization of the signaling capacities of the novel gp130-like cytokine receptor

The gp130-like receptor (GPL) is a recently cloned member of the family of type I cytokine receptors. The name reflects its close relationship to gp130, the common receptor subunit of the interleukin (IL)-6-type cytokines. Indeed, the recently proposed ligand for GPL, IL-31, is closely related to the IL-6-type cytokines oncostatin M, leukemia inhibitory factor, and cardiotrophin-1. The second signal transducing receptor for IL-31 seems to be the oncostatin M receptor beta (OSMRbeta). The present study characterizes in depth the molecular mechanisms underlying GPL-mediated signal transduction. GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identify tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of GPL (GPL(745)) as the major STAT5- and STAT3-activating sites, respectively. Additionally, we demonstrate Jak1 binding to GPL and its activation in heteromeric complexes with the OSMRbeta but also in a homomeric receptor complex. Most interesting, unlike OSMRbeta and gp130, GPL is insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of recruitment of the tyrosine phosphatase SHP-2 or the adaptor protein Shc to the cytoplasmic domain of GPL.     

Oncostatin M: signal transduction and biological activity

Oncostatin M (OSM) is a multifunctional cytokine produced by activated T lymphocytes and monocytes that is structurally and functionally related to the subfamily of cytokines known as the IL-6-type cytokine family. OSM shares properties with all members of this family of cytokines, but is most closely related structurally and functionally to LIE OSM acts on a wide variety of cells and elicits diversified biological responses in vivo and in vitro which suggest potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. OSM and LIF can bind to the same functional receptor complex (LIF-receptor beta and gp130 heteromultidimers) and thus mediate overlapping spectra of biological activities. There is a second specific beta receptor that binds OSM with high affinity and also involves the subunit gp130. The two receptors for OSM can be functionally different and be coupled to different signal transduction pathways. OSM-specific receptors are expressed in a wide variety of cell types and do not possess an intrinsic tyrosine kinase domain, but the JAK/STAT tyrosine kinase pathway mediates signal transduction.     

The carboxyl-terminal domains of gp130-related cytokine receptors are necessary for suppressing embryonic stem cell differentiation. Involvement of STAT3

Cell type-specific responses to the leukemia inhibitory factor (LIF)/interleukin 6 cytokine family are mediated by dimerization of the LIF receptor alpha-chain (LIFRalpha) with the signal transducer gp130 or of two gp130 molecules followed by activation of the JAK/STAT and Ras/mitogen-activated protein kinase cascades. In order to dissect the contribution of gp130 and LIFRalpha individually, chimeric molecules consisting of the extracellular domain of the granulocyte colony stimulating factor receptor (GCSF-R) and various mutant forms of the cytoplasmic domains of gp130 or LIFRalpha were expressed in embryonic stem (ES) cells to test for suppression of differentiation, or in a factor-dependent plasma cytoma cell line to assess for induction of proliferation. Carboxyl-terminal domains downstream of the phosphatase (SHP2)-binding sites were dispensable for mitogen-activated protein kinase activation and the transduction of proliferative signals. Moreover, carboxyl-terminal truncation mutants which lacked intact Box 3 homology domains showed decreased STAT3 activation, failed to induce Hck kinase activity and suppress ES cell differentiation. Moreover, STAT3 antisense oligonucleotides impaired LIF-dependent inhibition of differentiation. Substitution of the tyrosine residue within the Box 3 region of the GSCF-R abolished receptor-mediated suppression of differentiation without affecting the transduction of proliferative signals. Thus, distinct cytoplasmic domains within the LIFRalpha, gp130, and GCSF-R transduce proliferative and differentiation suppressing signals.     

Non-redundant signal transduction of interleukin-6-type cytokines. The adapter protein Shc is specifically recruited to rhe oncostatin M receptor

The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr(861) of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr(861) is essential for activation of ERK1/2 and for full activation of the alpha(2)-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.     

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.     

Molecular and functional characterization of a soluble form of oncostatin M/interleukin-31 shared receptor

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulin-like domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.     

Oncostatin M receptor-mediated signal transduction is negatively regulated by SOCS3 through a receptor tyrosine-independent mechanism

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.     

Signaling mechanisms through gp130: A model of the cytokine system

The interleukin-6 cytokine family plays roles in a wide variety of tissues and organs, including the immune hematopoietic and nervous systems. Gp130 is a signal-transducing subunit shared by the receptors for the IL-6 family of cytokines. The binding of a ligand to its receptor induces the dimerization of gp 130, leading to the activation of JAK tyrosine kinase and tyrosine phosphorylation of gpl30. These events lead to the activation of multiple signal-transduction pathways, such as the STAT, Ras-MAPK and PI-3 kinase pathways whose activation is controlled by distinct regions of gp130. We propose a model showing that the outcome of the signal transduction depends on the balance or interplay among the contradictory signal transduction pathways that are simultaneously generated through a cytokine receptor in a given target cell.     

Soluble glycoprotein 130 (gp130) attenuates OSM- and LIF-induced cartilage proteoglycan catabolism

Oncostatin M (OSM) and leukaemia inhibitory factor (LIF) exhibit pleiotropic biological activities and share many structural and genetic features. The two cytokines bind with high affinity to the same receptor (LIF/OSM receptor), which consists of the LIF receptor alpha chain (LIFRalpha) and the signal transduction unit gp130. A soluble form of the beta chain of the receptor complex called soluble gp130 (sgp130) has been cloned. In this study, we sought to determine whether recombinant sgp130 or anti-gp130 Ab could attenuate the resorption of proteoglycans induced by OSM and LIF in articular cartilage explants. The results show that at high concentrations sgp130 is capable of attenuating both LIF and OSM mediated resorption. In contrast, anti-gp130 Ab selectively inhibited the stimulation of proteoglycan (PG) release by OSM, albeit minimally. The failure of anti-gp130 to attenuate LIF stimulated PG resorption may be due to the normal interaction of LIF with LIFRalpha and unfettered heterodimerization of LIFRalpha with gp130 in the presence of the antibody. The results indicate that sgp130 and anti-gp130 can modulate cartilage PG metabolism in vitro. Whether sgp130 may have therapeutic activity in models of arthritis or indeed in arthritic diseases remains to be determined.     

Cloning and Characterization of a Specific Receptor for Mouse Oncostatin M

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor β [LIFRβ], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRβ and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.     

Characterization of the rat oncostatin m receptor complex which resembles the human, but differs from the murine cytokine receptor

Evaluation of a pathophysiological role of the interleukin-6-type cytokine oncostatin M (OSM) for human diseases has been complicated by the fact that mouse models of diseases targeting either OSM or the OSM receptor (OSMR) complex cannot fully reflect the human situation. This is due to earlier findings that human OSM utilizes two receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) (type I) and gp130/OSMR (type II), both with wide expression profiles. Murine OSM on the other hand only binds to the gp130/OSMR (type II) receptor complex with high affinity. Here, we characterize the receptor usage for rat OSM. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant and stably transfected Ba/F3 cells) we can clearly show that rat OSM surprisingly utilizes both, the type I and type II receptor complex, therefore mimicking the human situation. Furthermore, it displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the Jak/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, rat disease models would allow evaluation of the relevance of OSM for human biology.     

Determinants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.     

gp130 dimerization in the absence of ligand: preformed cytokine receptor complexes

It is established that cytokine receptors signal after ligand binding as homo- or hetero-dimers in heteromeric complexes, but it is unclear, when dimerization occurs. To investigate gp130 dimerization, we performed co-precipitation experiments with the endogenous cytokine receptors gp130 and leukemia inhibitory factor receptor (LIF-R) and with gp130 variants carrying two different C-terminal peptide tags. Furthermore, fluorescence resonance energy transfer (FRET) was employed to detect dimerization of two fluorescent-tagged gp130 variants. Confocal laser scanning microscopy was used for FRET detection in live cells. gp130 and LIF-R could be coprecipitated in the absence of ligand. The interaction, however, was intensified by the addition of LIF. Similar results were obtained with the gp130 variants and confirmed by FRET analysis in live cells. The present study clearly demonstrates the existence of preformed but inactive gp130/LIF-R hetero- and gp130/gp130 homo-dimers. The addition of ligand enhanced the respective dimer formation and was required for signal transduction.