Contributions of Leukemia Inhibitory Factor Receptor and Receptor to Signal Transduction in Heterodimeric Complexes with This information is current as of September 23, 2021. Heike M. Hermanns, Simone Radtke, Claude Haan, Hildegard Schmitz-Van de Leur, Jan Tavernier, Peter C. Heinrich and Iris Behrmann J Immunol 1999; 163:6651-6658; ; http://www.jimmunol.org/content/163/12/6651 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Contributions of Leukemia Inhibitory Factor Receptor and Oncostatin M Receptor to Signal Transduction in Heterodimeric Complexes with Glycoprotein 1301

Heike M. Hermanns,2* Simone Radtke,2* Claude Haan,* Hildegard Schmitz-Van de Leur,* Jan Tavernier,† Peter C. Heinrich,* and Iris Behrmann3*

Leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M (OSM) lead to heterodimeriza- tion of LIF receptor (LIFR) or the OSM-specific receptor (OSMR) with glycoprotein (gp) 130, the common receptor subunit for IL-6-type cytokines. Thereby intracellular signaling via Janus kinases (Jaks) and STAT transcription factors is initiated. We investigated the contributions of LIFR and OSMR to signal transduction in the context of heterodimers with gp130. Chimeric

receptors based on the extracellular parts of the IL-5R ␣- and ␤-chains were generated, allowing the induced heterodimerization Downloaded from of two different cytoplasmic tails. Our studies demonstrate that upon heterodimerization with the gp130 cytoplasmic region, the cytoplasmic parts of both LIFR and OSMR were critical for activation of an acute phase protein promoter in HepG2 hepatoma cells. The membrane-proximal region of LIFR or OSMR was crucial for the ability of such receptor complexes to induce DNA binding of STAT1 and STAT3 in COS-7 cells. Membrane-distal regions of LIFR and OSMR contributed to STAT activation even in the absence of gp130 STAT recruitment sites. We further show that the Janus kinases Jak1 and Jak2 constitutively associated with receptor constructs containing the cytoplasmic part of LIFR, OSMR, or gp130, respectively. Homodimers of the LIFR or http://www.jimmunol.org/ OSMR cytoplasmic regions did not elicit responses in COS-7 cells but did in HepG2 cells and in MCF-7 breast carcinoma cells. Thus, in spite of extensive functional similarities, differential signaling abilities of gp130, LIFR, and OSMR may become evident in a cell-type-specific manner. The Journal of Immunology, 1999, 163: 6651–6658.

he family of IL-6-type cytokines consists of IL-6, IL-11, IL-6-type cytokines have in part overlapping functions: e.g., IL-6, LIF, oncostatin M (OSM),4 ciliary neurotrophic factor, LIF, as well as OSM are able to induce the synthesis of acute phase T and cardiotrophin-1. They play important roles in the im- proteins in hepatocytes (4–7). All three cytokines induce macrophage mune system, during hematopoiesis and inflammation, for neuro- differentiation of mouse promyelocytic M1 cells (8–10). This func- by guest on September 23, 2021 genesis, heart development, bone remodeling, and reproduction tional redundancy can be explained by the shared use of the receptor (for recent reviews, see Refs. 1 and 2). Binding to their receptors subunit gp130. Whereas IL-6 and IL-11 induce homodimerization induces dimerization of the signal-transducing receptor chains. As- of gp130, the other IL-6-type cytokines lead to heterodimerization of sociated tyrosine kinases of the Janus family (Jak1, Jak2, Tyk2) gp130 with the LIF receptor (LIFR) or the OSM-specific receptor become activated and in turn phosphorylate specific tyrosine res- (OSMR) (1, 2). Human, but not murine, OSM can signal via a LIFR/ idues within the receptor chains. Thereby they create docking sites gp130 heterodimer in addition to the OSMR/gp130 receptor complex for proteins with matching Src homology 2 (SH2) domains, such (11, 12). Apart from exerting these redundant effects, each cytokine is as the tyrosine phosphatase SH2 domain-containing protein phos- additionally endowed with specific functions: e.g., LIF plays an im- phatase 2 (SHP-2) and transcription factors of the STAT family portant role for blastocyst implantation (13) and in activation of the (mainly STAT3 and STAT1). Subsequently, STATs also become hypothalamic-pituitary-adrenal axis during stress and inflammation tyrosine phosphorylated, form homo- or heterodimers, and trans- (14). OSM, secreted predominantly by activated macrophages and T locate into the nucleus, where they bind to specific enhancer ele- cells, is also produced by AIDS-associated Kaposi sarcoma cells and ments to induce expression of target (3). mediates their proliferation (15, 16). Thus, the restricted pattern of cytokine expression and the distribution of the ligand binding ␣ re- ceptors may explain cytokine-specific effects. Moreover, differences *Department of Biochemistry, Rheinisch-Westfa¨lische Technische Hochschule in signaling of homo- vs heterodimers have been noted: e.g., overex- † Aachen, Germany; and Department of Medical Protein Chemistry, Flanders Inter- pression of the transcription factor SCL inhibits the LIF- and OSM-, university Institute for Biotechnology, University of Ghent, Ghent, Belgium but not the IL-6-mediated induction of M1 cell differentiation (17). Received for publication May 18, 1999. Accepted for publication October 7, 1999. Dexamethasone inhibits the induction of the thiostatin by LIF 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 but not by IL-6 (18). The molecular basis underlying these differential with 18 U.S.C. Section 1734 solely to indicate this fact. signaling events is currently unknown. 1 This work was supported by the Deutsche Forschungsgemeinschaft (Bonn) and by To analyze OSMR and LIFR functions in transfected cells in- Fonds der Chemischen Industrie (Frankfurt). dependently of endogenous receptors, various groups have taken 2 H.M.H. and S.R. contributed equally to this study. advantage of chimeric receptor systems: G-CSF-induced ho- 3 Address correspondence and reprint requests to Dr. I. Behrmann, Department of modimerization of the cytoplasmic region of the OSMR and mu- Biochemistry, Rheinisch-Westfa¨lische Technische Hochschule Aachen, Pau- welsstrasse 30, 52074 Aachen, Germany. E-mail address: [email protected] tants thereof led to STAT activation and gene induction in hepa- 4 Abbreviations used in this paper: OSM, oncostatin M; OSMR, OSM receptor; LIFR, toma cells. Some differences to the signal transduction of a gp130/ LIF receptor; Epo, ; EpoR, Epo receptor. OSMR heterodimer were noted (19). Similarly, ligand-induced

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 6652 CONTRIBUTIONS OF LIFR AND OSMR TO SIGNAL TRANSDUCTION homodimerization of the cytoplasmic regions of LIFR led to bio- MCF-7 cells, XhoI/BamHI fragments comprising the cDNA encoding the logical responses in some (19–23) but not all cells (24, 25), in spite various receptor constructs were cloned into expression vector pCAGGS of the fact that the LIFR is only known to signal in combination (31) digested with XhoI and BglII. Expression plasmids pRK5-Jak1 and pRK5-Jak2 were kindly provided by Dr. I. Kerr (London, England) and Dr. with gp130. J. N. Ihle (Memphis, TN). An expression vector for Tyk2 was generated by The aim of this study was to investigate the contribution of the inserting Tyk2 cDNA (generously provided by Dr. S. Pellegrini, Paris, LIFR and OSMR to signal transduction in heterodimeric complexes France) into vector pSVL (Pharmacia, Piscataway, NJ). The STAT3 ex- with gp130. We therefore used a receptor system based on the extra- pression vector has been described (30). cellular parts of the IL-5R ␣- and ␤-chains (26) which allows the Cell culture and transient transfections directed formation of heterodimers, thereby mimicking the proposed Simian monkey kidney cells (COS-7) and the human breast carcinoma cell natural receptor complexes (gp130/LIFR or gp130/OSMR). line MCF-7 were maintained in DMEM, human hepatoma cells (HepG2) in Using this system, we demonstrate that the membrane-proximal DMEM/F12 medium supplemented with 10% FCS, 100 mg/L streptomy- regions of LIFR or OSMR are crucial for signal transduction in the cin, and 60 mg/L penicillin. Approximately 1.5 ϫ 107 COS-7 cells were heterodimeric receptor complex. Only one cytoplasmic tail has to transiently transfected with 10–20 ␮g plasmid DNA using the DEAE- contain STAT recruitment sites and these can be contributed by dextrane method. Briefly, cells were incubated for 90 min in 7.5 ml FCS- free medium containing the plasmid DNA, 6 ␮l chloroquine (100 mM), either gp130, LIFR, or OSMR. We further show constitutive as- and 60 ␮l DEAE-dextrane (50 mg/ml) for 90 min, avoiding gas exchange. sociation of Janus kinases not only with gp130, but also with the Afterward, cells were incubated for 1 min in PBS containing 10% DMSO. LIFR and OSMR. Moreover, evidence is provided for distinct sig- After extensive washing and cultivation for additional 48–72 h, cells were naling characteristics of gp130, LIFR, or OSMR apart from their harvested. HepG2 and MCF-7 cells were transfected with 20 ␮g plasmid DNA functional homologies. using the calcium-phosphate method as described previously (32). Downloaded from Materials and Methods Flow cytometry Reagents COS-7 cells were released from the dishes using PBS/10 mM EDTA, washed, and resuspended in cold PBS supplemented with 5% FCS and Restriction enzymes and T4-DNA ligase were obtained from Boehringer 0.1% sodium azide (PBS/azide). Cells (5 ϫ 105–1 ϫ 106) were incubated Mannheim (Mannheim, Germany) or AGS (Heidelberg, Germany), and with 1 ␮g/ml of either the monoclonal anti-IL-5R␣ Ab (16-4) or anti-IL- protease inhibitors were obtained from Sigma (Munich, Germany). DMEM 5R␤ Ab (S-16) for 30 min at 4°C. After washing the cells with cold PBS/ http://www.jimmunol.org/ and DMEM/F12 were purchased from Life Technologies (Eggenstein, Ger- azide, they were incubated with a 1/100 dilution of PE-conjugated goat many) and FCS from Seromed (Berlin, Germany). Human IL-5 was ex- Ј anti-mouse IgG-F(ab )2 (Dianova) for 30 min at 4°C. Cells were washed pressed in Sf9 insect cells and purified as described previously (27). Hu- again with cold PBS/azide, resuspended in PBS/azide, and analyzed by man recombinant erythropoietin was kindly provided by Drs. J. Burg and flow cytometry using a FACScalibur (Becton Dickinson, Mountain View, K.-H. Sellinger (Boehringer Mannheim, Penzberg, Germany). For flow cy- CA) equipped with a 488-nm argon laser. tometry, the mAb 16-4 specific for the human IL-5R ␣-chain (J. Van der Heyden and J. Tavernier, unpublished results) and the mAb S-16 specific EMSA for the human ␤c-chain (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Polyclonal antisera against human ␤c and Jak2 were purchased from Forty-eight to 72 h after transfection, COS-7 cells were starved for 4–6 h Santa Cruz Biotechnology, and Tyk2-specific antiserum was purchased and stimulated with 80 ng IL-5/ml for 30 min or with 7 U Epo/ml for 15 from Transduction Laboratories (Lexington, KY). Antiserum against Jak1 min. Nuclear extracts were prepared as described (33). Protein concentra- by guest on September 23, 2021 was a kind gift from Dr. A. Ziemicki (Bern, Switzerland). PE-labeled goat tions were measured with the Bio-Rad protein assay (Bio-Rad, Richmond, Ј CA). A double-stranded mutated SIE oligonucleotide from the c-fos pro- anti-mouse IgG-F(ab )2 was obtained from Dianova (Hamburg, Germany). moter (m67SIE: 5Ј-GAT CCG GGA GGG ATT TAC GGG AAA TGC Plasmid construction TG-3Ј) was labeled by filling in 5Ј protruding ends with the Klenow en- zyme using [␣-32P]dATP (3000 Ci/mmol; 10 mCi/ml). Nuclear extracts The construction of the chimeras IL-5R/gp130, IL-5R/gp130-B1/2, IL- containing 5–10 ␮g protein were incubated with about 10 fmol (10,000 5R␤/⌬cyt, IL-5R␤/gp130⌬box1, IL-5R/LIFR, and cpm) of probe in gel shift incubation buffer (10 mM HEPES (pH 7.8), 1 (EpoR)/gp130 have been described in previous studies (26, 28–30). The ␮ ␮ mM EDTA, 5 mM MgCl2, 10% glycerol, 5 M DTT, 0.7 M PMSF, 0.1 mutant IL-5R␤/LIFR-B1/2 was cloned by deleting a CelII/BamHI-frag- mg/ml of poly(dI-dC), and 1 mg/ml BSA) for 10 min at room temperature. ment from the expression plasmid pSVL-IL-5R␣/LIFR and exchanging the The protein-DNA complexes were separated on a 4.5% polyacrylamide gel sequence encoding the extracellular part of IL-5R␣ by the sequence en- containing 7.5% glycerol in 0.25-fold TBE (200 mM Tris, 166 mM boric coding the corresponding fragment of IL-5R␤. Because of the cloning pro- acid, 2 mM EDTA, adjusted to pH 8.3) at 20 V/cm for 4 h. Gels were fixed cedure, three additional amino acids (Ile-Glu-Thr) were added after posi- in a water solution of 10% methanol and 10% acetic acid for 30 min, dried, 931 tion Glu , followed by the termination codon. The IL-5R/OSMR and autoradiographed. Data were further analyzed with a Storm 840 Phos- constructs were generated by RT-PCR using a RT-PCR from Boehr- phorImager (Molecular Dynamics, Sunnyvale, CA). inger Mannheim. The sense primer annealing upstream of the sequence encoding the OSMR transmembrane region contained an in-frame EcoRI Immunoprecipitations and Western blotting site at its 5Ј end. Thus, the OSMR sequence starting with Thr727 is pre- ceded by a phenylalanine residue. The antisense primer contained a BamHI Forty-eight to 72 h after transfection, COS-7 cells were washed twice with site next to the stop codon. Total RNA (1 ␮g) isolated from OSM-sensitive PBS, scraped off the dish, and lysed in BRIJ96-lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% BRIJ96, 1 mM EDTA, 10 mM NaF, 1 mM human A375 melanoma cells was used for RT. The resulting cDNA was ␮ ␮ further amplified by PCR using the same primers as before according to the Na3VO4, 1 mM PMSF, 5 g/ml aprotinin, and 5 g/ml leupeptin) for 30 manufacturer’s instructions. The resulting OSMR fragment was inserted min on ice. Cell lysates were centrifuged at 14,000 rpm for 10 min. The ␣ supernatants were used for immunoprecipitation of the receptor chimeras into EcoRI/BamHI digested pSVL-based expression vectors for IL-5R / ␤ gp130 and IL-5R␤/gp130, respectively. A series of chimeric receptors en- using the anti-IL-5R Ab S-16. After overnight incubation at 4°C, immune coding truncated cytoplasmic OSMR domains was generated by PCR using complexes were collected on protein A-Sepharose during a 60-min incu- 3Ј oligonucleotides incorporating in-frame termination codons followed by bation, washed twice with washing buffer (as lysis buffer, but with only the recognition site for BamHI. OSMR⌬1, OSMR⌬2, OSMR⌬3, and 0.1% BRIJ96), and boiled for 5 min in Laemmli buffer at 95°C. The pro- OSMR-B1/2 retain 191, 162, 153, and 65 amino acids of the OSMR cy- teins were separated by 7.5% SDS-PAGE, followed by electroblotting onto toplasmic tail, respectively. The resulting PCR products were inserted into a polyvinylidene difluoride membrane (PALL, Dreieich, Germany). West- the EcoRI- and BamHI-digested expression plasmid pSVL-IL5R␤/OSMR. ern blot analysis was conducted with the indicated Abs and the enhanced The integrity of all constructs was verified by DNA sequence analyses chemiluminescence kit (Amersham, Arlington Heights, IL) according to using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Norwalk, CT). the manufacturer’s instructions. EpoR/LIFR and EpoR/OSMR constructs were generated by exchanging Reporter gene assays the EcoRI/BamHI fragment of pSVL-EpoR/gp130 encoding the transmem- ␣ Ϫ ϩ brane and intracellular region of gp130 by fragments encoding the corre- pGL3 2M-215Luc contains the promoter region 215 to 8 of the rat ␣ sponding regions of LIFR and OSMR. For transfection of HepG2 and 2-macroglobulin gene upstream of the luciferase-encoding sequence of The Journal of Immunology 6653

FIGURE 2. The cytoplasmic regions of LIFR and OSMR contribute to

signaling in heterodimeric complexes with gp130. HepG2 cells were trans- Downloaded from fected with expression plasmids encoding ␣/gp130 and the indicated ␤ ␣ chimera along with an 2-macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-5 (80 ng/ml) for 24 h or left untreated. Luciferase activity of lysates was nor- malized to the activity of coexpressed ␤-galactosidase. The fold inductions (relative to untreated cells) of three to four independent experiments (mean

Ϯ SD) are presented. http://www.jimmunol.org/ FIGURE 1. Schematic representation of chimeric receptors used in this study. Tyrosine residues in the intracellular regions are indicated as black lines. The box1 and box2 regions are depicted as hatched boxes. The do- ␣ ϩ ␣ ␤ IL-5 stimulation of both receptor combinations ( /gp130 main structure of the extracellular region of the IL-5 and receptors and ␤ ␣ ϩ ␤ the EpoR is schematically shown. Conserved cysteine residues (black /LIFR, /gp130 /OSMR), a strong induction of a luciferase ␣ lines) and WSXWS boxes (black boxes) of cytokine binding modules are reporter gene under the control of the 2-macroglobulin promoter indicated. was detectable (Fig. 2). This induction was comparable to that observed upon induced homodimerization of gp130 cytoplasmic tails (␣/gp130 ϩ ␤/gp130). ␣/gp130 did not elicit luciferase ex-

plasmid pGL3 (Promega, Madison, WI) (32). For reporter gene assays, pression when dimerized with ␤/⌬cyt, a receptor chimera devoid by guest on September 23, 2021 HepG2 or MCF-7 cells were transfected with 8 ␮g of luciferase reporter of a cytoplasmic region (Fig. 2). This indicates that the cytoplas- construct, 4 ␮gof␤-galactosidase control plasmid pCH110 (Pharmacia), mic parts of LIFR and OSMR crucially contribute to signaling in ␮ and 4 g of each receptor expression vector. Twenty-four hours after trans- ␣ fection, cultures were subdivided, and after another 12-h recovery period, heterodimeric complexes with /gp130. treated for 24 h with 80 ng/ml IL-5. Luciferase assays were performed using the Promega luciferase assay system. The values in each experimen- Membrane-proximal and -distal regions of the LIFR contribute tal series were normalized to ␤-galactosidase activity. to STAT activation in a heterodimer with gp130 To achieve higher transfection efficiencies, we switched to COS-7 Results cells which enabled us to monitor surface expression of receptor Heterodimerization of the LIFR or OSMR cytoplasmic region chimeras by flow cytometry and to measure DNA binding of with the one of gp130 leads to gene induction in hepatoma cells STATs by EMSA. When cells expressing ␣/gp130 ϩ ␤/gp130 To study the contribution of the LIFR and OSMR to signal trans- were stimulated with IL-5, STAT1 became activated as revealed duction in the respective heterodimeric complexes with gp130, we by the appearance of a respective band in gel shift assays (Fig. 3A, generated chimeric receptor constructs as shown in Fig. 1. They left panel; see Ref. 26). However, the STAT DNA binding activity consist of the extracellular domains of IL-5R␣, IL-5R␤,orthe induced by heterodimerization of the cytoplasmic parts of LIFR EpoR, and the transmembrane and intracellular parts of gp130, and gp130 was less pronounced relative to the one induced by LIFR, or OSMR. Previous studies have shown that IL-5 binding gp130 homodimerization, although LIFR and gp130 chimeras affinities of the IL-5R chimeras were not influenced by deletion or were equally well expressed as shown by FACS analysis (Fig. 3B). exchange of the cytoplasmic regions (26). No signaling was ob- DNA binding activity of endogenous STAT3 is hardly detectable served when only a single chimera, ␣/gp130 or ␤/gp130, was ex- in COS-7 cells (Fig. 3A, left panel; see Refs. 26 and 30), possibly pressed, demonstrating that signaling-competent receptor com- due to a low expression level of STAT3. Upon overexpression of plexes have to contain both the ␣- and the ␤-chain chimeras (26). STAT3, IL-5 stimulation of the various receptor combinations re- Therefore, this system is suitable to delineate the relative contri- sulted in a slower migrating protein-DNA complex (Fig. 3A, right butions of LIFR or OSMR to signal transduction within a het- panel) which could be supershifted with anti-STAT3 Abs (data not erodimer with gp130. shown). Also, in the presence of overexpressed STAT3, both In human HepG2 hepatoma cells, LIF and OSM, as well as IL-6, gp130/LIFR heterodimeric receptor complexes elicited weaker are able to induce the expression of genes encoding acute phase STAT responses compared with the ␣/␤-gp130 homodimer. proteins (5, 7, 34). These cells are therefore an appropriate system To further delineate the contributions of LIFR to signal trans- to test whether the chimeric receptor constructs ␤/LIFR and duction, we took advantage of two chimeras (␣/gp130-B1/2; ␤/OSMR were functional, i. e., able to transduce signals upon ␤/LIFR-B1/2) that lack the cytoplasmic regions distal from heterodimerization with ␣/gp130. After transient transfection and box1/2. Interestingly, a receptor combination of ␣/gp130-B1/2 6654 CONTRIBUTIONS OF LIFR AND OSMR TO SIGNAL TRANSDUCTION

to signaling in combination with gp130: whereas a receptor com- bination in which only one gp130 cytoplasmic chain was present (␣/gp130 ϩ ␤/⌬cyt) did not elicit STAT activation, the combina- tion with the membrane-proximal region of the LIFR (␣/gp130 ϩ ␤/LIFR-B1/2) resulted in a STAT signal (Fig. 4, middle panel), which was somewhat weaker than the one elicited by the full- length receptor combination (shown for comparison in the right panel of Fig. 4). Taken together, these results suggest that the LIFR has the po- tential to exert the same signal-transducing functions as gp130, albeit to a lesser extent (Fig. 3).

Membrane-proximal and -distal regions of the OSMR contribute to STAT activation in a heterodimer with gp130 We next investigated the signaling potential of OSMR in combi- nation with gp130 using IL-5R chimeras containing the transmem- brane and cytoplasmic region of OSMR (see Fig. 1). As observed for the LIFR chimera, activation of STAT1 upon IL-5 stimulation Downloaded from of heterodimeric OSMR/gp130 chimeras was weaker than the one observed upon homodimerization of gp130 cytoplasmic tails (Fig. 5A, left panel). However, STAT3, when overexpressed, was more strongly activated by the OSMR/gp130 heterodimer (Fig. 5A, right panel), indicating that the OSMR might be a very potent activator

of STAT3. http://www.jimmunol.org/ When surface expression was monitored, it became evident that chimeras containing the cytoplasmic region of the OSMR were expressed at a lower level than gp130 or LIFR chimeras (data not FIGURE 3. Induced heterodimerization of the cytoplasmic parts of the shown). C-terminal truncations of the OSMR (OSMR⌬1, LIFR and gp130 leads to STAT activation. A, COS-7 cells were transfected OSMR⌬2, OSMR⌬3) resulted in higher expression levels as dem- with expression plasmids encoding the IL-5R ␣ and ␤ chimeras as indi- onstrated by Western blot analysis (Fig. 5B). However, induced cated. Right panel, Cells were cotransfected with 5 ␮g of a STAT3 ex- heterodimerization of ␣/gp130 with truncated ␤/OSMR constructs pression vector. Three days after transfection, cells were stimulated with did not lead to increased STAT signals. Indeed, loss of the C- IL-5 (80 ng/ml) for 30 min or left untreated before nuclear extracts were terminal regions of OSMR resulted in less intense EMSA bands by guest on September 23, 2021 prepared. EMSAs were performed using the m67SIE probe. The bands (Fig. 5C) and reduced ␣ -macroglobulin promoter activation in resulting from STAT1 or STAT3 homodimers are indicated. B, Surface 2 expression of chimeric receptors. A fraction of the transfectants was ana- HepG2 cells (data not shown) compared with the combination of lyzed by flow cytometry using Abs directed against IL-5R␣ and IL-5R␤. full-length receptors. This indicates a function of the OSMR mem- The filled histograms depict cells expressing the receptor chimeras, brane-distal part for signal transduction which is further demon- whereas the open histograms represent mock-transfected cells. strated in Fig. 6 (left panel): a receptor combination devoid of the gp130 STAT recruitment sites (␣/gp130-B1/2 ϩ ␤/OSMR) is still able to elicit STAT activation. Apart from the C-terminal region, with full-length ␤/LIFR was able to induce a STAT signal, membrane-proximal sequences of the OSMR also play an impor- whereas a combination in which only the membrane-proximal tant role for signal transduction via an OSMR/gp130 heterodimer: parts of the LIFR and gp130 were present was not functional (Fig. combination of the full-length cytoplasmic part of gp130 with the 4, left panel). Thus, the C-terminal region of the LIFR can mediate OSMR box1/2 region resulted in a strong STAT response (Fig. 6, STAT activation in combination with gp130-B1/2, suggesting that right panel). Thus, also the OSMR, as shown for the LIFR above, the LIFR tyrosine motifs serve as STAT recruitment sites. More- provides functional motifs necessary for signaling in combination over, also the membrane-proximal region of the LIFR contributes with gp130.

FIGURE 4. Both the membrane-proximal and -distal parts of the LIFR contribute to sig- naling in a heterodimeric complex with gp130. COS-7 cells were transfected with expression plasmids encoding STAT3 and IL-5R ␣ and ␤ chimeras as indicated. Three days after trans- fection, cells were stimulated with IL-5 (80 ng/ ml) for 30 min or left untreated before nuclear extracts were prepared. EMSAs were per- formed using the m67SIE probe. The bands re- sulting from STAT3 homodimers are indicated. The Journal of Immunology 6655

FIGURE 6. Both the membrane-proximal and -distal parts of the OSMR contribute to signaling in a heterodimeric complex with gp130. COS-7 cells were transfected with expression plasmids encoding STAT3 and IL-5R ␣ and ␤ chimeras as indicated. Three days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min or left untreated before nuclear extracts were prepared. EMSAs were performed using the m67SIE Downloaded from probe. The bands resulting from STAT3 homodimers are indicated.

␤/OSMR⌬1 (Fig. 7, upper panels). Binding of Tyk2 could be shown for ␤/gp130 and ␤/LIFR (Fig. 7, lower panel), but not con- ␤ ⌬ vincingly for /OSMR 1 (data not shown). Jaks did not bind to a http://www.jimmunol.org/ gp130 construct with a deletion in the membrane-proximal region including the box1 motif (Fig. 7). Moreover, no unspecific precip- itation was observed in the absence of receptor chimeras (data not shown). Western blots of cellular lysates demonstrate comparable expression levels of each kinase within one set of experiments. Although the OSMR construct was expressed to a lower degree (see above), approximately equivalent amounts of receptor were precipitated, indicating that the anti-IL-5R␤ Ab was used in lim-

FIGURE 5. Induced heterodimerization of the cytoplasmic parts of iting concentrations (Fig. 7). by guest on September 23, 2021 OSMR with gp130 leads to STAT activation. A, COS-7 cells were trans- fected with expression plasmids encoding the IL-5R ␣ and ␤ chimeras as The signaling capacity of homodimerized LIFR or OSMR indicated. Right panel, Cells were cotransfected with 5 ␮g of a STAT3 cytoplasmic parts depends on the cellular context expression vector. Three days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min or left untreated before nuclear extracts were Although LIFR and OSMR are only known to participate in het- prepared. EMSAs were performed using the m67SIE probe. The bands erodimeric receptor complexes with gp130, artificial homodimer- resulting from STAT1 and STAT3 homodimers are indicated. B, Succes- ization of the respective cytoplasmic regions by chimeric receptors sive C-terminal deletion of the cytoplasmic part of the OSMR leads to elicited cellular responses in several studies (19–24) but not in increased expression levels. Three days after transfection of COS-7 cells others (24, 25). By combining the respective IL-5R ␣ and ␤ chi- with expression constructs encoding ␤/OSMR or the various deletion con- meras, we studied the effects of homodimerization of the cytoplas- structs, lysates were prepared and analyzed in a Western blot developed mic parts of the LIFR or OSMR. Compared with IL-5R/gp130 with a polyclonal antiserum against human IL-5R␤. C, Successive C-ter- homodimers, both the LIFR and the OSMR homodimers yielded minal truncation of the OSMR cytoplasmic region results in decreased only marginal activation of endogenous STAT1 or overexpressed STAT activation upon heterodimerization with the cytoplasmic region of STAT3 in COS-7 cells (Fig. 8A). In addition, we replaced the gp130. COS-7 cells were transfected with expression plasmids encoding ␣/gp130 and ␤/OSMR or successively truncated derivatives thereof as in- extracellular region of the IL-5R by the one of the EpoR, a ho- dicated. Lower panel, Cells were cotransfected with 5 ␮g of STAT3 ex- modimer, which has been successfully applied for construction of pression vector. EMSAs were performed as described in A. hybrid receptors (30, 32, 35). However, also the Epo-induced ho- modimerization of the cytoplasmic parts of LIFR or OSMR elic- ited only marginal STAT responses, whereas homodimerization of EpoR/gp130 led to a strong signal (Fig. 8B). In striking contrast, Jak1 and Jak2 associate with OSMR, LIFR, and gp130 homodimerized cytoplasmic parts of LIFR and OSMR were able Janus kinases are known to associate with the membrane-proximal to induce reporter gene activation in HepG2 hepatoma and MCF-7 region of cytokine receptors. IL-6-type cytokines lead to the acti- breast carcinoma cells (Fig. 8C). Thus, the signaling ability of vation of Janus kinases Jak1, Jak2, and Tyk2. Therefore, we com- LIFR and OSMR homodimers seems to depend on the cellular pared the three receptor subunits gp130, LIFR, and OSMR with context. respect to their ability to associate with Jaks. Finally, the IL-5R chimeras enabled us to study the effects of a COS-7 cells were cotransfected with expression vectors encod- heterodimerization of the LIFR and OSMR cytoplasmic parts. ing ␤/gp130, ␤/LIFR, or ␤/OSMR⌬1 (we used the deletion con- Even this combination of receptors led to gene activation in hep- struct to achieve higher expression levels, see Fig. 5B) and one of atoma and in breast carcinoma cells (Fig. 8C), underlining the the three Janus kinases. After lysis under mild conditions, Jak1 and functional homology of these three receptors involved in signaling Jak2 could be coimmunoprecipitated with ␤/gp130, ␤/LIFR, and of all IL-6-type cytokines. 6656 CONTRIBUTIONS OF LIFR AND OSMR TO SIGNAL TRANSDUCTION Downloaded from http://www.jimmunol.org/

FIGURE 8. The signaling capacity of homodimerized LIFR and OSMR cytoplasmic parts depends on the cellular context. A and B, COS-7 cells were transfected with expression plasmids encoding the IL-5R ␣ and ␤ or EpoR chimeras as indicated. Right panels, Cells were cotransfected with 5 ␮g of a STAT3 expression vector. Three days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min or with Epo (7 U/ml) for 15 min or left untreated before nuclear extracts were prepared. EMSAs were per- formed using the m67SIE probe. Bands resulting from STAT3 or STAT1 by guest on September 23, 2021 homodimers are indicated. C, Reporter gene assays. HepG2 or MCF-7 cells ␣ were transfected with the indicated expression plasmids along with an 2- macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-5 (80 ng/ml) for 24 h or left untreated. Luciferase activity of lysates was normalized to the activity of coexpressed ␤-galactosidase. The -fold inductions (relative to untreated cells) of three to four independent experiments (mean Ϯ SD) are presented, except for heterodimerization of LIFR and OSMR cytoplasmic parts in HepG2 cells (two experiments).

erodimeric gp130/LIFR and gp130/OSMR complexes. One major finding is that the membrane-proximal cytoplasmic regions of LIFR and OSMR play a crucial role for signaling. These regions comprise box1/box2 sequences conserved in gp130 as well as in other cytokine receptors and are crucial for recruitment of Janus FIGURE 7. Jak association with gp130, LIFR, and OSMR. COS-7 cells kinases (3, 36). In fact, Janus kinases associate with all three re- were cotransfected with expression constructs encoding one receptor chi- ceptors: we could demonstrate binding of Jak1 and Jak2 to OSMR mera (␤/gp130, ␤/LIFR, ␤/OSMR⌬1, or ␤/gp130⌬box1) and one Janus sequences, whereas Jak1, Jak2, and Tyk2 coprecipitated with re- kinase (Jak1, Jak2, or Tyk2), respectively. After lysis of the cells, immu- ceptors containing intracellular regions of gp130 and LIFR. It is noprecipitates of the receptor and aliquots of cellular lysates were sepa- believed that the presence of (at least) two receptor-associated Jaks rated by SDS-PAGE and blotted onto polyvinylidene difluoride mem- is required for initiation of downstream signaling events upon cy- branes. Western blots were developed with Abs against Jak1, Jak2, Tyk2, tokine-induced receptor dimerization (3, 37). Thus, it is very likely or IL-5R␤ as indicated. It should be noted that in other experiments the somewhat reduced binding of Jak1 to ␤/LIFR (compared with ␤/gp130) that LIFR or OSMR provide the respective “second” was not apparent. in heterodimeric complexes with gp130. Our data extend previous studies demonstrating association of LIFR with Jak1 and Jak2 (38) and association of gp130 with Jak1, Jak2, and/or Tyk2 (38–41). Our findings are in accordance with other reports showing that the Discussion presence of the membrane-proximal region of LIFR heterodimer- In the present study, we analyzed the contributions of the cyto- ized with gp130 is sufficient to mediate gp130 tyrosine phosphor- plasmic parts of LIFR or OSMR to signal transduction in het- ylation, STAT activation, gene induction, or hematopoietic cell The Journal of Immunology 6657 proliferation and differentiation (19, 20, 42). However, in contrast OSMR homodimers, as well as corresponding receptors containing to our results, a truncated OSMR retaining the membrane-proximal the extracellular part of EpoR, elicited only very weak STAT signals. 60 amino acid residues (comprising box1/2) was found to be un- Several authors have investigated the signaling ability of homodimer- able to support STAT activation and gene induction upon dimer- ized LIFR cytoplasmic chains with partly contradictory results: sig- ization with gp130 in rat hepatoma cells (19). The usage of dif- naling could be demonstrated in hepatoma and neuroblastoma cells ferent cell types, receptor constructs, and experimental readouts (20), embryonic stem cells (24, 43), and COS-1 cells (21) when chi- may explain this apparent discrepancy. meric receptors were used that homodimerized upon stimulation with Dimerization of the membrane-proximal region of gp130 with their respective ligands (G-CSF, neurotrophin-3, or epidermal growth the full-length cytoplasmic parts of LIFR or OSMR but not with factor). On the other hand, G-CSFR/LIFR constructs did not elicit the respective truncated box1/2 constructs leads to STAT activa- signaling in M1 promyelocytic cells or BAF/03 pre-B cells (24). Sim- tion (Figs. 4 and 6). This finding demonstrates that 1) the mem- ilarly, Nakamura et al. (25) did not observe signaling upon GM-CSF- brane-distal parts of the LIFR or OSMR provide critical STAT induced homodimerization of two LIFR cytoplasmic parts in embry- recruitment sites, 2) that they are functional even in the absence of onic stem cells, although GM-CSF-induced heterodimers of the the gp130 membrane-distal region, and 3) that only a single chain cytoplasmic parts of LIFR and gp130 elicited a response. However, within such a receptor dimer has to be equipped with STAT re- GM-CSF-induced homodimers of LIFR cytoplasmic regions were cruitment sites. We show for the first time that only the membrane- able to induce differentiation in M1 and WEHI-3B promyelocytic proximal region of gp130 is sufficient to allow signal transduction cells (23). Thus, the cellular background and maybe even the subline when dimerized with full-length LIFR or OSMR cytoplasmic used, the transfected hybrid receptors, and the different experimental parts. Their ability to activate STATs in the absence of gp130 readouts may affect the results of such studies so that it is difficult to Downloaded from STAT recruitment sites was so far demonstrated only upon artifi- draw general conclusions. It will be of interest to find out to what cially induced homodimerization of the cytoplasmic parts of LIFR degree the apparent cell-type specific signaling of chimeric LIFR ho- or OSMR (19–23). The contribution of OSMR STAT recruitment modimers might be due to differences in expression of the various modules was also implicated by our finding that successive C- Jaks or in their activation profile. The signaling ability of homodimers terminal truncations of the OSMR led to decreased STAT re- of the OSMR cytoplasmic region has been addressed so far only in

␣ http://www.jimmunol.org/ sponses in COS-7 cells (Fig. 5B) and 2-macroglobulin promoter one study (19). induction in HepG2 cells (data not shown) when dimerized with Our study provides evidence that the three signal-transducing full-length cytoplasmic gp130. Intriguingly, however, a C-terminal polypeptide chains gp130, LIFR, and OSMR relevant for signaling truncation of only 36 amino acids of the OSMR totally abolished of IL-6-type cytokines have common properties: they critically the ability of the OSMR/gp130 complex to activate STATs in contribute to signaling via heterodimeric gp130/LIFR or gp130/ Hep3B hepatoma cells (19). In accordance with the study by Ku- OSMR receptor complexes and both membrane-proximal and ropatwinski et al. (19), we could demonstrate that C-terminal de- membrane-distal parts are involved. Their functional homology letions of the OSMR lead to enhanced protein expression. Future could further be demonstrated by their interchangeability: even studies will aim at the identification of the molecular basis under- enforced heterodimerization of the cytoplasmic parts of LIFR and lying this phenomenon. OSMR elicited reporter gene activation in HepG2 hepatoma and by guest on September 23, 2021 For the chimeric LIFR/gp130 heterodimer, we observed a lower MCF-7 breast carcinoma cells (Fig. 8C). Apart from common STAT1- and STAT3-activating potential in spite of equal expres- functions, the differential signaling ability of homodimeric recep- sion levels of the surface receptors (Fig. 3). It is very unlikely that tors in COS-7 cells points at distinct characteristics of gp130, this is due to a lower affinity for IL-5 since the affinity of the IL-5R LIFR, or OSMR, which possibly become evident only in certain does not seem to be influenced by the receptor’s intracellular re- cells. Redundant effects of IL-6-type cytokines have been attrib- gion: we have previously demonstrated that the affinity of IL-5R/ uted to the shared usage of gp130 as a common signal-transducing gp130 chimeras (such as the affinity conversion upon coexpression chain and the structural as well as functional similarity of gp130, of a IL-5R␤ chimera) is comparable to the one observed for the LIFR, and OSMR. Thus, many of the biological effects elicited by wild-type IL-5R complex in COS transfectants (26). Also, receptor a gp130 homodimer can be also observed for the LIFR/gp130 or complexes incompetent of signal transduction such as ␣/gp130 ϩ OSMR/gp130 heterodimer, if the corresponding ligand binding re- ␤/⌬cyt (see Figs. 4 and 6) or IL-5R/gp130 chimeras, fused down- ceptor chains are provided. However, several reports have pointed stream of the transmembrane region, bound IL-5 with normal high at differences in signal transduction events elicited from gp130/ affinity (Ref. 26; H. M. Hermanns, unpublished data). gp130 homodimers and gp130/LIFR or gp130/OSMR het- Overexpression of STAT3 in COS-7 cells revealed a difference erodimers which cannot be explained by differential receptor ex- in signaling via OSMR vs LIFR: coexpression of STAT3 signifi- pression (e.g., different preferences in STAT activation (19, 44, cantly increased the STAT signal elicited by heterodimeric gp130/ 45), differentiation of M1 transfectants (17) and PC-12 pheochro- OSMR chimeras relative to that induced by homodimers of the mocytoma cells (44), and proliferation vs growth inhibition of gp130 cytoplasmic part (Fig. 5), whereas the signal intensity of breast carcinoma cells (46–48)). Therefore, our receptor chimeras heterodimeric gp130/LIFR chimeras remained unchanged (Fig. 3). are promising tools to analyze the molecular basis of these differ- This suggests that the OSMR, compared with the LIFR, may be a ences in signal transduction via heterodimeric gp130/LIFR and more potent activator of STAT3, although it contains only two gp130/OSMR complexes. tyrosine modules corresponding to the consensus sequence for STAT3 activation YXXQ (30, 38): Y917 (YVSQ) and Y945 (YKMQ), whereas the LIFR contains three such motifs: Y981 Acknowledgments (YQPQ), Y1001 (YKPQ), and Y1028 (YRPQ). We thank Drs. L. Graeve, G. Mu¨ller-Newen, F. Schaper, and J. Kuhse for Cytokines signaling via homomeric LIFR or OSMR complexes critical reading of this manuscript. We are grateful to Dr. A. Ziemicki (devoid of gp130) are currently unknown. Nonetheless, IL-5 in- (Bern) for providing antiserum against Jak1. We thank Dr. I. M. Kerr duced homodimerization of the cytoplasmic regions of LIFR and (London), and Dr. S. Pellegrini (Paris) for providing the plasmids encoding OSMR elicited signals in HepG2 hepatoma and in MCF-7 breast Janus kinases. Human recombinant Epo was a generous gift from Boehr- carcinoma cells. In COS-7 cells, however, IL-5R/LIFR or IL-5R/ inger Mannheim. 6658 CONTRIBUTIONS OF LIFR AND OSMR TO SIGNAL TRANSDUCTION

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