Gp130 transducing protein was shown to be involved in the formation of the high affinity receptors for interleukin 6 (IL-6), interleukin-11 (IL-11), leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1. In the present study we have characterized the functional properties of antibodies directed against this protein and identified a group of monoclonal antibodies able to antagonize the biological activities of all the cytokines belonging to the IL-6 cytokine family. The B-R3 pan-blocking antibody weakly interfered with the binding of the radiolabeled ligands (with the exception of OSM, whose binding was abrogated in the presence of B-R3 monoclonal antibody) but inhibited the gp130 homodimerization or its association with gp190/leukemia inhibitory factor receptor, as well as the subsequent tyrosine phosphorylation events. In addition we identified antibodies that were able to neutralize only one single cytokine of the IL-6 family. This was the case for the B-K5 antibody, which antagonized the binding of OSM to gp130 but did not interfere with the signals provided by the related cytokines triggering the proliferation of the TF1 erythroleukemia cell line or the induction of haptoglobin synthesis in the HepG2 hepatoma cell line. Similarly, we also characterized two additional antibodies B-P8 and B-P4, which inhibited the TF1 cell proliferation observed in the presence of CNTF and IL-11, respectively. B-P8 antibody only faintly interfered with the binding of the gp130-ligands and might modulate the signal transduction pathways. This study indicates that in addition to functional site(s) required by the whole family of IL-6 type cytokines to transduce the signal insight the cell, specific cognate functional sites were recruited by OSM, CNTF, or IL-11.

Oncostatin M (OSM) mediates its bioactivities through two different heterodimer receptors. They both involve the gp130-transducing receptor, which dimerizes with either leukemia inhibitory receptor ␤ or with OSM receptor ␤ (OSMR␤) to generate, respectively, type I and type II OSM receptors. Co-precipitation of gp130-associated proteins, flow cytometry, polymerase chain reaction, and tyrosine phosphorylation analyses allowed the characterization of both types of OSM receptors expressed on the surface of different cell lines. It also allowed the detection of a large size protein, p250, that specifically associates to the type II OSM receptor components and that is tyrosine-phosphorylated after the activation peak of the gp130⅐OSMR␤ heterocomplex. The restricted expression of type I OSM receptor by the JAR choriocarcinoma cell line, and type II receptor by the A375 melanoma cell line, permitted the characterization of their signaling machineries. Both type I and type II OSM receptors activated Jak1, Jak2, and Tyk2 receptor-associated tyrosine kinases. The information is next relayed to the nucleus by the STAT3 transcriptional activator, which is recruited by both types of OSM receptors. In addition, STAT5b was specifically activated through the gp130⅐OSMR␤ type II heterocomplex.The signaling pathway differences observed between the common type I LIF/OSM receptor and the specific type II OSM receptor might explain some of the bioactivities specifically displayed by OSM.Oncostatin M (OSM) 1 is a multifunctional cytokine belonging to the interleukin-6 (IL-6) family and that shares many properties with those reported for LIF (1). Both OSM and LIF are able to inhibit the spontaneous differentiation of embryonic stem cells (2). They also induce the terminal differentiation of the M1 murine myeloid cell line (3). Like the other IL-6 family members, OSM has been shown to induce acute phase protein synthesis in hepatocytes (4). Beside these LIF-shared bioactivities, OSM displays some specific properties and can inhibit the growth of a variety of solid tumor cells (5) and triggers in vitro the proliferation of Kaposi's sarcoma-derived cell lines (6, 7). In addition, expression of an oncostatin M transgene in the early T cell lineage stimulates a dramatic accumulation of T cells in the mice lymph nodes (8).The redundancy of OSM and LIF biological properties is in part explained by the shared use of a common heterocomplex receptor composed of the gp130 signal transducing protein associated with the LIF receptor ␤ (LIFR␤) component (9, 10). Binding experiments have pointed out the existence of a second and different high affinity receptor for OSM (also referred to as type II OSM receptor) (9 -11). Type II OSM receptor complex binds OSM in a specific manner and is not recognized by LIF (9 -11). Type II receptor also involves a gp130-transducing component that associates with a second receptor subunit very recently isolated as OSM receptor ␤ (OSMR␤), which displays an apparent molecular mass of 180 kDa (12). Comparison of OSMR␤ t...

The recently cloned interleukin (IL)-11 displays many biological properties in common with those reported for IL-6. In order to analyze the nature and the functionality of the IL-11 receptor we developed a proliferative assay using the human multifactor-dependent cell line TF1. We showed that a blocking monoclonal antibody GPX7 raised against the gp130/IL-6 receptor transducing subunit was also able to inhibit the IL-11-triggered TF1 line proliferation. In addition, involvement of gp130 in IL-11 signaling was demonstrated by an induction of the transducing protein phosphorylation in response to IL-11, as observed for IL-6. In contrast, the blocking monoclonal antibody B-R6, which recognized the gp80/IL-6 binding subunit failed to interfere with the IL-11 proliferative signal in the TF1 cell line. Similarly, we did not observe any competition between IL-6 and IL-11 for a putative common binding site on the cell surface. These results suggest that the IL-11 binding component is different from the gp80/IL-6 receptor. In conclusion, IL-11, along with IL-6, leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor, belongs to the same family of cytokines, using gp130 as a transducing protein.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study we show that CT-1 activates its receptor expressed at the surface of a human neural cell line by recruiting gp130 and gp190/leukemia inhibitory factor receptor ␤, as shown by analyzing their tyrosine phosphorylation level. Neutralizing antibody directed against gp130 and reconstitution experiments performed in the COS-7 cell line demonstrate that gp130-gp190 heterocomplex formation is essential for CT-1 signaling. Analysis of the subsequent activation events revealed that CT-1 induces and utilizes Jak1-, Jak2-, and Tyk2-associated tyrosine kinases, which are in turn relayed by STAT-3 transcription factor. Cross-linking of iodinated CT-1 to the cell surface led to the identification of a third ␣ component in addition to gp130 and gp190, with an apparent molecular mass of 80 kDa. Removal of N-linked carbohydrates from the protein backbone of the ␣ component resulted in a protein of 45 kDa. Our results provide evidence that the CT-1 receptor is composed of a tripartite complex, a situation similar to the high affinity receptor for ciliary neurotrophic factor.

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