Interleukin-6 Signal Transducer -130 Mediates Oncostatin M Signaling*

M a multifunctional cytokine that is structurally and functionally related to interleukin (IL-6) and inhibitory factor (LIF). The specific for been (by cross-linking) to be a 150-kDa in a number of We examined the possible involvement of in OM binding and signaling. We demonstrate cross-linking,

Oncostatin M (OM),' a 28-kDa glycoprotein, is a newly characterized multifunctional cytokine (1, 2) which shares structural and functional homology to several cytokines including IL-6 and LIF (3,4). OM acts on a wide variety of cells and elicits diversified biological responses such as growth stimulation, growth inhibition (1,5), leukemic cell differentiation (3,6), acute phase protein induction (7), LDL receptor up-regulation (8), and cell-specific gene expression (6,7). OM * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: OM, oncostatin M; IL-6, interleukin-6; LIF, leukemia inhibitory factor; mAb, monoclonal antibody; TGF, transforming growth factor; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Hepes, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. appears to mediate its effects through a specific cellular receptor that has been characterized in a variety of cell lines to be a protein with an approximate molecular mass of 150 kDa (5-10). The biochemical and molecular nature of the OM receptor remains elusive. The overlapping biological effects of OM and IL-6 in many cellular systems, and the isolation of a mutant A375 human melanoma cell line that is resistant to only OM and IL-6, but not to other cytokines (such as TGF-P, IL-1, tumor necrosis factor-a and -P, and interferonr),* suggested that OM and IL-6 share a common component in their signal transduction pathways. Therefore, we investigated the involvement of the IL-6 signal transducer, gp130 (ll), in OM binding and signaling. We report conclusively that OM is the natural ligand for gp130 and also that gp130 is a necessary component of OM signaling machinery. Recently, Gearing et al. (12) reported that low affinity OM binding sites are expressed on COS-7 cells transfected with a truncated gp130 cDNA.

EXPERIMENTAL PROCEDURES
Cells and Reagents-The human melanoma cell line A375 (ATCC), the human lung carcinoma cell line H2981 (Bristol-Myers Squibb), and the African green monkey kidney cell line COS-7 (ATCC) were cultured in DMEM medium supplemented with 10% fetal bovine serum. GPX7, GPX22, and GPZ35 are mouse anti-human gp130 monoclonal antibodies3 Human recombinant oncostatin M was expressed in Chinese hamster ovary cell supernatants and purified by reverse-phase high performance liquid chromatography (13).
Cell Proliferation Assays-Cells were incubated overnight in 96well tissue culture plates (Costar) in DMEM medium (100 pl/well) containing 5% serum a t a concentration of 3000 cells/well. Cultures were re-fed and incubated with the same culture medium supplemented with various test factors. Three days later cells were incubated in medium containing 5-[1Z51]iodo-2'-deoxyuridine (0.1 pCi/well) for 6 h, washed with PBS, fixed in 95% methanol, and air-dried. Cell pellets were solubilized with 200 pl of 1 N sodium hydroxide and the amount of lZ5I incorporated into DNA was measured using a y radiation counter. The differences in counts/min incorporated between experimental and control cultures were used as the index for growth. Each data point represents the average from triplicate cultures.
Chemical Cross-linking-The confluent monolayers were washed once and removed from the culture plates by incubation with cell removal buffer (50 mM Hepes, pH7.4, 1 mM EDTA, 5 mM glucose, 5 mM KC1, 125 mM NaCl, 2 mg/ml bovine serum albumin, and 1 fig/ ml each leupeptin, aprotinin, and pepstatin A) for 10 min at 37 "C. The iodination of OM and cross-linking to cells was conducted as essentially described (9).
Immunoprecipitation-Approximately 1.7 X lo7 H2981 cells were cross-linked to lZ5I-OM and subsequently extracted with 1 ml of digitonin extraction buffer (11) for 5 h at 4 'C. The debris was removed by centrifugation at 7000 X g for 10 min, and the supernatants were then incubated with antibodies for 16 h. The immunoreactive complexes were then immunoprecipitated by protein A-Sepharose and analyzed by SDS-PAGE (9).
COS Cell Transfections and Binding Assay-COS-7 cells grown in tissue culture flaskettes (Nunc, Inc.) were transfected with plasmid DNA by the DEAE-dextran-chloroquine method (14). Forty-eight hours after transfection, cells were incubated with lZ5I-OM (2000 Ci/ mM) for 2 h at room temperature and then extensively washed. The autoradiographic analysis of transfected cells was performed as described (15). tissue culture plates were incubated for 2 days in DMEM containing 5% fetal bovine serum and various amounts of purified recombinant OM ( A ) , IL-6 ( E ) , and TGF-P ( C ) with and without 4 pg/ml of anti-gp130 mAbs or control mouse mAb. A mixture of anti-gpl30 mAbs GPX7, GPX22, and GPZ35 was used in this and subsequent experiments. The control mouse mAb was anti-CD4 mAb. Cells were pulsed with ['251]iododeoxyurine (0.1 pCi/ml) for an additional 6 h. The amount of radioactivity incorporated into DNA was determined, and the data was normalized to the percent of the growth of the culture relative to cells not treated with the factors. W, medium alone, 0, + anti-gpl30 mAbs, El, + control mouse mAb.

RESULTS AND DISCUSSION
To investigate the possible involvement of the IL-6 signal transducer, gp130, in OM signaling, we examined the effects of monoclonal antibodies (mAbs) against the / 3 subunit of the IL-6 receptor (gp130) on the growth inhibition of A375 cells by OM, IL-6, and TGF-P. We observed that anti-gpl30 mAbs (4 pg/ml), but not isotype-matched irrelevant murine mAb (control mouse mAb), were able to prevent the growth inhibition caused by OM and IL-6 but were unable to prevent the growth inhibition caused by TGF-P (Fig. 1). The results suggested that gp130 is involved in transducing OM signals, and could be a component of the OM receptor.
Neither IL-6 nor LIF bind to the human lung carcinoma cell line H2981; however, these cells possess 1-3 x lo4 binding sites/cell for OM and express mRNA encoding the gp130 p r~t e i n .~ In order t o clarify the role of gp130 in OM-receptor interactions we examined the ability of anti-gpl30 mAbs to block the binding of radiolabeled OM to H2981 cells. Binding J. Liu, unpublished data. of lZ5I-OM to H2981 was completely blocked by 10 pg/ml of anti-gpl30 mAbs but was unaffected by the control mouse mAb (Fig. 2). This suggested that OM binds directly to gp130.
Ligand-receptor cross-linking studies with lZ5I-OM and H2981 cells were carried out to biochemically characterize the OM receptor on these cells. SDS-PAGE analysis of OMbinding proteins on HZ981 cells showed one major species with an apparent molecular mass of 150 kDa (180-kDa labeled complex minus 30-kDa OM) and another minor species with an apparent molecular mass of 300 kDa. The cross-linking of OM to these two molecules was inhibited by unlabeled OM or anti-gpl30 mAbs, but not by IL-6 or control mouse mAb (Fig. 3). At present, the relationship between these two OM binding species is not clear. However, we believe that the 300-kDa species could be a receptor complex that includes OM, gp130, and another subunit or the dimer of gp130. It is possible that the 150-kDa protein cross-linked to OM is not gp130 but is instead a closely related molecule that shares a common binding epitope for the anti-gpl30 mAbs. However, we think this is highly unlikely because four different monoclonal anti-gp130 mAbs (AM64, AM66, GPX7, GPX22) individually blocked the cross-linking of OM to the 150-kDa protein.
To further demonstrate the relationship between the OM receptor and gp130, lZ5I-OM was cross-linked to its receptor on H2981 cells, and the cells were subsequently washed, solubilized with a lysis buffer containing digitonin, and then immunoprecipitated with the anti-gpl30 mAbs or with the control mouse mAb. Analysis of this material by SDS-PAGE showed that anti-gpl30 mAbs were able to immunoprecipitate the radiolabeled protein of 180 kDa (150-kDa + 30-kDa OM). k D a p r o t e i n with a n t i -g p l 3 0 mAbs. H2981 cells (1.5 X lo7) were cross-linked with 1 nM 12sI-OM, then solubilized with digitonin lysis buffer. After spinning briefly in a microcentrifuge, the supernatant was divided equally into three parts and immunoprecipitated with medium alone, anti-gpl30 mAbs, or control mouse mAh respectively, and analyzed by SDS-PAGE. Experimental details were as described (12). This radiolabeled protein was not immunoprecipitated by the control mouse mAb (Fig. 4). To illustrate the direct interaction between OM and the p subunit of the IL-6 receptor, COS cells were transfected with a mammalian expression vector containing a full-length cDNA encoding gp130. Two days post-transfection, the COS cell transfectants grown on glass slides were incubated with "'I-OM (ranging in concentration from 0.1 to 5 nM), fixed, and dipped in photographic emulsion. After a 48-h exposure, the slides were developed and examined by phase contrast and dark field microscopy. Slides containing COS cells transfected with the plasmid carrying the gp130 cDNA displayed positive cells as identified by the presence of autoradiographic grains. The binding of '*'I-OM to the COS cell transfectants was blocked by excess unlabeled OM or anti-gpl3O mAbs but was not blocked by unlabeled IL-6 or the control mouse mAb (Fig. 5).
COS cell transfectants expressing gp130 were cross-linked to '*'I-OM. This material was analyzed by SDS-PAGE and compared with that obtained from mock transfectants and H2981 cells cross-linked with l2'1-OM. Fig. 6A shows that '*'I-OM cross-linked protein migrated with an apparent molecular mass of 150 kDa in all cells; however, the intensity of signal was much higher in gp130 transfectants than in mock transfectants. Most importantly, all the cross-linked proteins in COS cells were competed out by anti-gpl30 mAbs (Fig. 6B).
These data, again, strongly suggest that the 150-kDa protein cross-linked to '*'I-OM is gp130. The 150-kDa band from   COS cells ran slightly diffused as compared with that of H2981, which may be due to differences in glycosylation of gp130 molecules in the two cell types.
Recently, Gearing et al. (12) reported that OM binds to COS-7 cells transfected with a cDNA encoding a truncated form of gp130 with an affinity of 7.7 X lo-' M, which is significantly lower than the low affinity OM binding expressed by H2981 and COS-7 cells (& = 8 f 2 X 10"' M). 4 Studies with COS cell transfectants expressing a full-length gp130 protein show that the complete gp130 protein binds OM with the same affinity as the low affinity OM binding receptor (data not shown), suggesting that the cytoplasmic domain of gp130 modulates the binding affinity of the extracellular domain of gp130. We identified the nature of the OM reactive molecule and demonstrated that OM is the natural ligand for gp130 and that gp130 mediates OM signaling.
Although the OM-gpl30 interaction is necessary for OMelicited biological responses it might not be sufficient for initiating OM signals. It is very likely that the OM receptor, like other cytokine receptors, is a dimer or oligomer consisting of gp130 and other subunit(s), which associates with gp130 to generate the functional receptor. There is, however, no experimental evidence for the existence of the putative p subunit of OM receptor. Nevertheless, the following facts suggest the contribution of another nonbinding subunit in OM signaling: ( a ) gp130 is widely expressed in a variety of cells, but most of the hematopoietic cells do not respond to OM; ( b ) BAF.BO3 cells transfected with gp130 cDNA fail to respond to OM under the conditions where IL-6 is able to transduce mitogenic signals'; ( c ) OM does not bind to B9 cells but binds to LIF receptor transfected B9 cells with an affinity lower than the binding to native OM receptor (16); ( d ) the majority of receptors for other members of the cytokine super family are dimers. Multiple receptor subunits and shared receptor subunits could provide a rational basis for the pleiotrophy and redundancy of OM function. The OM-gpl3O interaction represents a unique system where gp130, the signal transducer and affinity converter for IL-6 and LIF, is the ligand binder for OM. Cytokines IL-3, IL-5, and granulocyte macrophagecolony stimulating factor also share a common signal transducer named KH97 (17)(18)(19)(20). All of the ligands tested so far have failed to bind KH97, although future studies might reveal ' T. Taga, unpublished data. the ligand for KH97. Recently, OM has been shown to be the major growth factor for Kaposi's sarcoma-derived cells whose growth is also stimulated by IL-6 and other cytokines (21,22). Since gp130 is a shared and essential component of IL-6-, OM-, and LIF-signaling pathways, the interruption of gpl30-mediated signals by anti-gpl30-neutralizing mAbs or antisense gp130 RNA may be useful inhibitors of the growth of Kaposi's sarcoma cells in vitro and in uiuo.