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.     

IL-6 signal transduction and its physiological roles: the signal orchestration model

Interleukin (IL)-6 is a pleiotropic cytokine that not only affects the immune system, but also acts in other biological systems and many physiological events in various organs. In a target cell, IL-6 can simultaneously generate functionally distinct or sometimes contradictory signals through its receptor complex, IL-6Rα and gp130. One good illustration is derived from the in vitro observations that IL-6 promotes the growth arrest and differentiation of M1 cells through gp130-mediated STAT3 activation, whereas the Y759/SHP-2-mediated cascade by gp130 stimulation has growth-enhancing effects. The final physiological output can be thought of as a consequence of the orchestration of the diverse signaling pathways generated by a given ligand. This concept, the signal orchestration model, may explain how IL-6 can elicit proinflammatory or anti-inflammatory effects, depending on the in vivo environmental circumstances. Elucidation of the molecular mechanisms underlying this issue is a challenging subject for future research. Intriguingly, recent in vivo studies indicated that the SHP-2-binding site- and YXXQ-mediated pathways through gp130 are not mutually exclusive but affect each other: a mutation at the SHP-2-binding site prolongs STAT3 activation, and a loss of STAT activation by gp130 truncation leads to sustained SHP-2/ERK MAPK phosphorylation. Although IL-6/gp130 signaling is a promising target for drug discovery for many human diseases, the interdependence of each signaling pathway may be an obstacle to the development of a nonpeptide orally active small molecule to inhibit one of these IL-6 signaling cascades, because it would disturb the signal orchestration. In mice, a consequence of the imbalanced signals causes unexpected results such as gastrointestinal disorders, autoimmune diseases, and/or chronic inflammatory proliferative diseases. However, lessons learned from IL-6 KO mice indicate that IL-6 is not essential for vital biological processes, but a significant impact on disease progression in many experimental models for human disorders. Thus, IL-6/gp130 signaling will become a more attractive therapeutic target for human inflammatory diseases when a better understanding of IL-6 signaling, including the identification of the conductor for gp130 signal transduction, is achieved.     

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.     

TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.     

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.     

A novel oncostatin M-inducible gene OIG37 forms a gene family with MyD118 and GADD45 and negatively regulates cell growth.

Oncostatin M (OSM) is a member of the IL-6 family cytokines that use gp130 as a common signal transducer and exhibits both growth stimulatory as well as growth inhibitory activity depending on the cells. To analyze the mechanism of OSM function, we isolated immediate early responsive genes upon OSM stimulation. Here we describe the novel OSM-inducible gene OIG37 that is related to MyD118 and GADD45. The MyD118 gene has been described as an immediate early gene induced by IL-6 in M1 monocytic cells, and GADD45 was identified as a gene induced by UV or gamma-ray irradiation. Both are considered to function in growth arrest and/or DNA repair. Although the expression of OIG37, MyD118, and GADD45 was rather ubiquitous, it was differentially regulated. As the gp130 mutant defective for activating the STAT3 pathway showed the reduced induction of OIG37 by cytokine stimulation and expression of dominant negative STAT3 inhibited the induction of OIG37 by OSM, STAT3 is involved in OIG37 induction by IL-6 family cytokines. To examine the function of OIG37, we expressed it in NIH3T3 and IL-3-dependent BaF3 cells and found that OIG37 suppressed cell growth without any evidence of apoptosis. Whereas both MyD118 and OIG37 suppressed cell growth in both cell lines, suppression by OIG37 was more efficient than by MyD118. Immunoprecipitation experiments indicated that OIG37 associates with p21, a cyclin-dependent kinase inhibitor, and proliferating cell nuclear antigen.     

STAT3 is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rbeta2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.     

gp130-Dependent signalling pathway is not enhanced in gp130 transgenic heart after LIF stimulation

Activation of gp130 transduces a hypertrophic signal in the heart, but it is not clear whether signalling through gp130 is enhanced when gp130 is overexpressed in vivo. We generated gp130 transgenic mice (TG) and examined the activation of signalling pathways downstream of gp130 in the hearts. The tyrosine phosphorylation of gp130 was enhanced, the phosphorylation of STAT3 and ERK (extracellular signal regulated kinase) 1/2 was increased and induction of the beta-myosin heavy chain (MHC) gene was observed in TG hearts without significant phenotypic changes. Intravenous administration of leukaemia inhibitory factor (LIF) induced tyrosine phosphorylation of STAT3 and ERK 1/2 and expression of c-fos and beta-MHC mRNAs in wild-type littermates' (WT) hearts. However, enhancement of STAT3 and ERK 1/2 phosphorylation or augmented mRNA expressions was not observed in TG hearts after LIF stimulation. Next, STAT-induced STAT inhibitor (SSI) mRNA expression was examined. The expression of SSI-1, SSI-2, and SSI-3 mRNAs was significantly augmented in TG hearts after LIF stimulation. These results indicate that overexpressed gp130 does not always enhance downstream signals in the hearts and suggest that the SSI family plays a role in the regulation of the gp130-dependent signalling pathway in the hearts.