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The Journal of Immunology, 1999, 163: 6651-6658.
Copyright © 1999 by The American Association of Immunologists

Contributions of Leukemia Inhibitory Factor Receptor and Oncostatin M Receptor to Signal Transduction in Heterodimeric Complexes with Glycoprotein 1301

Heike M. Hermanns2,*, Simone Radtke2,*, Claude Haan*, Hildegard Schmitz-Van de Leur*, Jan Tavernier{dagger}, Peter C. Heinrich* and Iris Behrmann3,*

* Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Germany; and {dagger} Department of Medical Protein Chemistry, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M (OSM) lead to heterodimerization 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 {alpha}- and ß-chains were generated, allowing the induced heterodimerization 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The family of IL-6-type cytokines consists of IL-6, IL-11, LIF, oncostatin M (OSM),4 ciliary neurotrophic factor, and cardiotrophin-1. They play important roles in the immune system, during hematopoiesis and inflammation, for neurogenesis, heart development, bone remodeling, and reproduction (for recent reviews, see Refs. 1, 2). Binding to their receptors induces dimerization of the signal-transducing receptor chains. Associated tyrosine kinases of the Janus family (Jak1, Jak2, Tyk2) become activated and in turn phosphorylate specific tyrosine residues within the receptor chains. Thereby they create docking sites for proteins with matching Src homology 2 (SH2) domains, such as the tyrosine phosphatase SH2 domain-containing protein phosphatase 2 (SHP-2) and transcription factors of the STAT family (mainly STAT3 and STAT1). Subsequently, STATs also become tyrosine phosphorylated, form homo- or heterodimers, and translocate into the nucleus, where they bind to specific enhancer elements to induce expression of target genes (3).

IL-6-type cytokines have in part overlapping functions: e.g., IL-6, LIF, as well as OSM are able to induce the synthesis of acute phase proteins in hepatocytes (4, 5, 6, 7). All three cytokines induce macrophage differentiation of mouse promyelocytic M1 cells (8, 9, 10). This functional redundancy can be explained by the shared use of the receptor subunit gp130. Whereas IL-6 and IL-11 induce homodimerization of gp130, the other IL-6-type cytokines lead to heterodimerization of gp130 with the LIF receptor (LIFR) or the OSM-specific receptor (OSMR) (1, 2). Human, but not murine, OSM can signal via a LIFR/gp130 heterodimer in addition to the OSMR/gp130 receptor complex (11, 12). Apart from exerting these redundant effects, each cytokine is additionally endowed with specific functions: e.g., LIF plays an important role for blastocyst implantation (13) and in activation of the hypothalamic-pituitary-adrenal axis during stress and inflammation (14). OSM, secreted predominantly by activated macrophages and T cells, is also produced by AIDS-associated Kaposi sarcoma cells and mediates their proliferation (15, 16). Thus, the restricted pattern of cytokine expression and the distribution of the ligand binding {alpha} receptors may explain cytokine-specific effects. Moreover, differences in signaling of homo- vs heterodimers have been noted: e.g., overexpression of the transcription factor SCL inhibits the LIF- and OSM-, but not the IL-6-mediated induction of M1 cell differentiation (17). Dexamethasone inhibits the induction of the thiostatin gene by LIF but not by IL-6 (18). The molecular basis underlying these differential signaling events is currently unknown.

To analyze OSMR and LIFR functions in transfected cells independently of endogenous receptors, various groups have taken advantage of chimeric receptor systems: G-CSF-induced homodimerization of the cytoplasmic region of the OSMR and mutants thereof led to STAT activation and gene induction in hepatoma cells. Some differences to the signal transduction of a gp130/OSMR heterodimer were noted (19). Similarly, ligand-induced homodimerization of the cytoplasmic regions of LIFR led to biological responses in some (19, 20, 21, 22, 23) but not all cells (24, 25), in spite of the fact that the LIFR is only known to signal in combination with gp130.

The aim of this study was to investigate the contribution of the LIFR and OSMR to signal transduction in heterodimeric complexes with gp130. We therefore used a receptor system based on the extracellular parts of the IL-5R {alpha}- and ß-chains (26) which allows the directed formation of heterodimers, thereby mimicking the proposed natural receptor complexes (gp130/LIFR or gp130/OSMR).

Using this system, we demonstrate that the membrane-proximal regions of LIFR or OSMR are crucial for signal transduction in the heterodimeric receptor complex. Only one cytoplasmic tail has to contain STAT recruitment sites and these can be contributed by either gp130, LIFR, or OSMR. We further show constitutive association of Janus kinases not only with gp130, but also with the LIFR and OSMR. Moreover, evidence is provided for distinct signaling characteristics of gp130, LIFR, or OSMR apart from their functional homologies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Restriction enzymes and T4-DNA ligase were obtained from Boehringer Mannheim (Mannheim, Germany) or AGS (Heidelberg, Germany), and protease inhibitors were obtained from Sigma (Munich, Germany). DMEM and DMEM/F12 were purchased from Life Technologies (Eggenstein, Germany) and FCS from Seromed (Berlin, Germany). Human IL-5 was expressed in Sf9 insect cells and purified as described previously (27). Human recombinant erythropoietin was kindly provided by Drs. J. Burg and K.-H. Sellinger (Boehringer Mannheim, Penzberg, Germany). For flow cytometry, the mAb 16-4 specific for the human IL-5R {alpha}-chain (J. Van der Heyden and J. Tavernier, unpublished results) and the mAb S-16 specific for the human ßc-chain (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Polyclonal antisera against human ßc and Jak2 were purchased from Santa Cruz Biotechnology, and Tyk2-specific antiserum was purchased from Transduction Laboratories (Lexington, KY). Antiserum against Jak1 was a kind gift from Dr. A. Ziemicki (Bern, Switzerland). PE-labeled goat anti-mouse IgG-F(ab')2 was obtained from Dianova (Hamburg, Germany).

Plasmid construction

The construction of the chimeras IL-5R/gp130, IL-5R/gp130-B1/2, IL-5Rß/{Delta}cyt, IL-5Rß/gp130{Delta}box1, IL-5R/LIFR, and erythropoietin receptor (EpoR)/gp130 have been described in previous studies (26, 28, 29, 30). The mutant IL-5Rß/LIFR-B1/2 was cloned by deleting a CelII/BamHI-fragment from the expression plasmid pSVL-IL-5R{alpha}/LIFR and exchanging the sequence encoding the extracellular part of IL-5R{alpha} by the sequence encoding the corresponding fragment of IL-5Rß. Because of the cloning procedure, three additional amino acids (Ile-Glu-Thr) were added after position Glu931, followed by the termination codon. The IL-5R/OSMR constructs were generated by RT-PCR using a RT-PCR kit from Boehringer Mannheim. The sense primer annealing upstream of the sequence encoding the OSMR transmembrane region contained an in-frame EcoRI site at its 5' end. Thus, the OSMR sequence starting with Thr727 is preceded by a phenylalanine residue. The antisense primer contained a BamHI site next to the stop codon. Total RNA (1 µg) isolated from OSM-sensitive 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 manufacturer’s instructions. The resulting OSMR fragment was inserted into EcoRI/BamHI digested pSVL-based expression vectors for IL-5R{alpha}/gp130 and IL-5Rß/gp130, respectively. A series of chimeric receptors encoding truncated cytoplasmic OSMR domains was generated by PCR using 3' oligonucleotides incorporating in-frame termination codons followed by the recognition site for BamHI. OSMR{Delta}1, OSMR{Delta}2, OSMR{Delta}3, and OSMR-B1/2 retain 191, 162, 153, and 65 amino acids of the OSMR cytoplasmic tail, respectively. The resulting PCR products were inserted into the EcoRI- and BamHI-digested expression plasmid pSVL-IL5Rß/OSMR. The integrity of all constructs was verified by DNA sequence analyses using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Norwalk, CT). EpoR/LIFR and EpoR/OSMR constructs were generated by exchanging the EcoRI/BamHI fragment of pSVL-EpoR/gp130 encoding the transmembrane and intracellular region of gp130 by fragments encoding the corresponding regions of LIFR and OSMR. For transfection of HepG2 and MCF-7 cells, XhoI/BamHI fragments comprising the cDNA encoding the various receptor constructs were cloned into expression vector pCAGGS (31) digested with XhoI and BglII. Expression plasmids pRK5-Jak1 and pRK5-Jak2 were kindly provided by Dr. I. Kerr (London, England) and Dr. J. N. Ihle (Memphis, TN). An expression vector for Tyk2 was generated by inserting Tyk2 cDNA (generously provided by Dr. S. Pellegrini, Paris, France) into vector pSVL (Pharmacia, Piscataway, NJ). The STAT3 expression vector has been described (30).

Cell culture and transient transfections

Simian monkey kidney cells (COS-7) and the human breast carcinoma cell line MCF-7 were maintained in DMEM, human hepatoma cells (HepG2) in DMEM/F12 medium supplemented with 10% FCS, 100 mg/L streptomycin, and 60 mg/L penicillin. Approximately 1.5 x 107 COS-7 cells were transiently transfected with 10–20 µg plasmid DNA using the DEAE-dextrane method. Briefly, cells were incubated for 90 min in 7.5 ml FCS-free medium containing the plasmid DNA, 6 µl chloroquine (100 mM), and 60 µl DEAE-dextrane (50 mg/ml) for 90 min, avoiding gas exchange. Afterward, cells were incubated for 1 min in PBS containing 10% DMSO. After extensive washing and cultivation for additional 48–72 h, cells were harvested.

HepG2 and MCF-7 cells were transfected with 20 µg plasmid DNA using the calcium-phosphate method as described previously (32).

Flow cytometry

COS-7 cells were released from the dishes using PBS/10 mM EDTA, washed, and resuspended in cold PBS supplemented with 5% FCS and 0.1% sodium azide (PBS/azide). Cells (5 x 105–1 x 106) were incubated with 1 µg/ml of either the monoclonal anti-IL-5R{alpha} Ab (16-4) or anti-IL-5Rß Ab (S-16) for 30 min at 4°C. After washing the cells with cold PBS/azide, they were incubated with a 1/100 dilution of PE-conjugated goat anti-mouse IgG-F(ab')2 (Dianova) for 30 min at 4°C. Cells were washed again with cold PBS/azide, resuspended in PBS/azide, and analyzed by flow cytometry using a FACScalibur (Becton Dickinson, Mountain View, CA) equipped with a 488-nm argon laser.

EMSA

Forty-eight to 72 h after transfection, COS-7 cells were starved for 4–6 h and stimulated with 80 ng IL-5/ml for 30 min or with 7 U Epo/ml for 15 min. Nuclear extracts were prepared as described (33). Protein concentrations were measured with the Bio-Rad protein assay (Bio-Rad, Richmond, CA). A double-stranded mutated SIE oligonucleotide from the c-fos promoter (m67SIE: 5'-GAT CCG GGA GGG ATT TAC GGG AAA TGC TG-3') was labeled by filling in 5' protruding ends with the Klenow enzyme using [{alpha}-32P]dATP (3000 Ci/mmol; 10 mCi/ml). Nuclear extracts containing 5–10 µg protein were incubated with about 10 fmol (10,000 cpm) of probe in gel shift incubation buffer (10 mM HEPES (pH 7.8), 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 5 µM DTT, 0.7 µM PMSF, 0.1 mg/ml of poly(dI-dC), and 1 mg/ml BSA) for 10 min at room temperature. The protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (200 mM Tris, 166 mM boric acid, 2 mM EDTA, adjusted to pH 8.3) at 20 V/cm for 4 h. Gels were fixed in a water solution of 10% methanol and 10% acetic acid for 30 min, dried, and autoradiographed. Data were further analyzed with a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitations and Western blotting

Forty-eight to 72 h after transfection, COS-7 cells were washed twice with 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 Na3VO4, 1 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin) for 30 min on ice. Cell lysates were centrifuged at 14,000 rpm for 10 min. The supernatants were used for immunoprecipitation of the receptor chimeras using the anti-IL-5Rß Ab S-16. After overnight incubation at 4°C, immune complexes were collected on protein A-Sepharose during a 60-min incubation, washed twice with washing buffer (as lysis buffer, but with only 0.1% BRIJ96), and boiled for 5 min in Laemmli buffer at 95°C. The proteins were separated by 7.5% SDS-PAGE, followed by electroblotting onto a polyvinylidene difluoride membrane (PALL, Dreieich, Germany). Western blot analysis was conducted with the indicated Abs and the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions.

Reporter gene assays

pGL3{alpha}2M-215Luc contains the promoter region -215 to +8 of the rat {alpha}2-macroglobulin gene upstream of the luciferase-encoding sequence of plasmid pGL3 (Promega, Madison, WI) (32). For reporter gene assays, HepG2 or MCF-7 cells were transfected with 8 µg of luciferase reporter construct, 4 µg of ß-galactosidase control plasmid pCH110 (Pharmacia), and 4 µg of each receptor expression vector. Twenty-four hours after transfection, cultures were subdivided, and after another 12-h recovery period, treated for 24 h with 80 ng/ml IL-5. Luciferase assays were performed using the Promega luciferase assay system. The values in each experimental series were normalized to ß-galactosidase activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heterodimerization of the LIFR or OSMR cytoplasmic region with the one of gp130 leads to gene induction in hepatoma cells

To study the contribution of the LIFR and OSMR to signal transduction in the respective heterodimeric complexes with gp130, we generated chimeric receptor constructs as shown in Fig. 1Go. They consist of the extracellular domains of IL-5R{alpha}, IL-5Rß, or the EpoR, and the transmembrane and intracellular parts of gp130, LIFR, or OSMR. Previous studies have shown that IL-5 binding affinities of the IL-5R chimeras were not influenced by deletion or exchange of the cytoplasmic regions (26). No signaling was observed when only a single chimera, {alpha}/gp130 or ß/gp130, was expressed, demonstrating that signaling-competent receptor complexes have to contain both the {alpha}- and the ß-chain chimeras (26). Therefore, this system is suitable to delineate the relative contributions of LIFR or OSMR to signal transduction within a heterodimer with gp130.



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  		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 domain structure of the extracellular region of the IL-5 {alpha} and ß receptors and the EpoR is schematically shown. Conserved cysteine residues (black lines) and WSXWS boxes (black boxes) of cytokine binding modules are indicated.

 
In human HepG2 hepatoma cells, LIF and OSM, as well as IL-6, are able to induce the expression of genes encoding acute phase proteins (5, 7, 34). These cells are therefore an appropriate system to test whether the chimeric receptor constructs ß/LIFR and ß/OSMR were functional, i. e., able to transduce signals upon heterodimerization with {alpha}/gp130. After transient transfection and IL-5 stimulation of both receptor combinations ({alpha}/gp130 + ß/LIFR, {alpha}/gp130 + ß/OSMR), a strong induction of a luciferase reporter gene under the control of the {alpha}2-macroglobulin promoter was detectable (Fig. 2Go). This induction was comparable to that observed upon induced homodimerization of gp130 cytoplasmic tails ({alpha}/gp130 + ß/gp130). {alpha}/gp130 did not elicit luciferase expression when dimerized with ß/{Delta}cyt, a receptor chimera devoid of a cytoplasmic region (Fig. 2Go). This indicates that the cytoplasmic parts of LIFR and OSMR crucially contribute to signaling in heterodimeric complexes with {alpha}/gp130.



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  		FIGURE 2. The cytoplasmic regions of LIFR and OSMR contribute to signaling in heterodimeric complexes with gp130. HepG2 cells were transfected with expression plasmids encoding {alpha}/gp130 and the indicated ß chimera along with an {alpha}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 ß-galactosidase. The fold inductions (relative to untreated cells) of three to four independent experiments (mean ± SD) are presented.

 
Membrane-proximal and -distal regions of the LIFR contribute to STAT activation in a heterodimer with gp130

To achieve higher transfection efficiencies, we switched to COS-7 cells which enabled us to monitor surface expression of receptor chimeras by flow cytometry and to measure DNA binding of STATs by EMSA. When cells expressing {alpha}/gp130 + ß/gp130 were stimulated with IL-5, STAT1 became activated as revealed by the appearance of a respective band in gel shift assays (Fig. 3GoA, left panel; see Ref. 26). However, the STAT DNA binding activity induced by heterodimerization of the cytoplasmic parts of LIFR and gp130 was less pronounced relative to the one induced by gp130 homodimerization, although LIFR and gp130 chimeras were equally well expressed as shown by FACS analysis (Fig. 3GoB). DNA binding activity of endogenous STAT3 is hardly detectable in COS-7 cells (Fig. 3GoA, left panel; see Refs. 26, 30), possibly due to a low expression level of STAT3. Upon overexpression of STAT3, IL-5 stimulation of the various receptor combinations resulted in a slower migrating protein-DNA complex (Fig. 3GoA, right panel) which could be supershifted with anti-STAT3 Abs (data not shown). Also, in the presence of overexpressed STAT3, both gp130/LIFR heterodimeric receptor complexes elicited weaker STAT responses compared with the {alpha}/ß-gp130 homodimer.



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  		FIGURE 3. Induced heterodimerization of the cytoplasmic parts of the LIFR and gp130 leads to STAT activation. A, COS-7 cells were transfected with expression plasmids encoding the IL-5R {alpha} and ß chimeras as indicated. Right panel, Cells were cotransfected with 5 µg of a STAT3 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 prepared. EMSAs were performed using the m67SIE probe. The bands resulting from STAT1 or STAT3 homodimers are indicated. B, Surface expression of chimeric receptors. A fraction of the transfectants was analyzed by flow cytometry using Abs directed against IL-5R{alpha} and IL-5Rß. The filled histograms depict cells expressing the receptor chimeras, whereas the open histograms represent mock-transfected cells.

 
To further delineate the contributions of LIFR to signal transduction, we took advantage of two chimeras ({alpha}/gp130-B1/2; ß/LIFR-B1/2) that lack the cytoplasmic regions distal from box1/2. Interestingly, a receptor combination of {alpha}/gp130-B1/2 with full-length ß/LIFR was able to induce a STAT signal, whereas a combination in which only the membrane-proximal parts of the LIFR and gp130 were present was not functional (Fig. 4Go, left panel). Thus, the C-terminal region of the LIFR can mediate STAT activation in combination with gp130-B1/2, suggesting that the LIFR tyrosine motifs serve as STAT recruitment sites. Moreover, also the membrane-proximal region of the LIFR contributes to signaling in combination with gp130: whereas a receptor combination in which only one gp130 cytoplasmic chain was present ({alpha}/gp130 + ß/{Delta}cyt) did not elicit STAT activation, the combination with the membrane-proximal region of the LIFR ({alpha}/gp130 + ß/LIFR-B1/2) resulted in a STAT signal (Fig. 4Go, 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. 4Go).



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  		FIGURE 4. Both the membrane-proximal and -distal parts of the LIFR contribute to signaling in a heterodimeric complex with gp130. COS-7 cells were transfected with expression plasmids encoding STAT3 and IL-5R {alpha} and ß 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 probe. The bands resulting from STAT3 homodimers are indicated.

 
Taken together, these results suggest that the LIFR has the potential to exert the same signal-transducing functions as gp130, albeit to a lesser extent (Fig. 3Go).

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 combination with gp130 using IL-5R chimeras containing the transmembrane and cytoplasmic region of OSMR (see Fig. 1Go). As observed for the LIFR chimera, activation of STAT1 upon IL-5 stimulation of heterodimeric OSMR/gp130 chimeras was weaker than the one observed upon homodimerization of gp130 cytoplasmic tails (Fig. 5GoA, left panel). However, STAT3, when overexpressed, was more strongly activated by the OSMR/gp130 heterodimer (Fig. 5GoA, right panel), indicating that the OSMR might be a very potent activator of STAT3.



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  		FIGURE 5. Induced heterodimerization of the cytoplasmic parts of OSMR with gp130 leads to STAT activation. A, COS-7 cells were transfected with expression plasmids encoding the IL-5R {alpha} and ß chimeras as indicated. Right panel, Cells were cotransfected with 5 µg of a STAT3 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 prepared. EMSAs were performed using the m67SIE probe. The bands resulting from STAT1 and STAT3 homodimers are indicated. B, Successive C-terminal deletion of the cytoplasmic part of the OSMR leads to increased expression levels. Three days after transfection of COS-7 cells with expression constructs encoding ß/OSMR or the various deletion constructs, lysates were prepared and analyzed in a Western blot developed with a polyclonal antiserum against human IL-5Rß. C, Successive C-terminal truncation of the OSMR cytoplasmic region results in decreased STAT activation upon heterodimerization with the cytoplasmic region of gp130. COS-7 cells were transfected with expression plasmids encoding {alpha}/gp130 and ß/OSMR or successively truncated derivatives thereof as indicated. Lower panel, Cells were cotransfected with 5 µg of STAT3 expression vector. EMSAs were performed as described in A.

 
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 shown). C-terminal truncations of the OSMR (OSMR{Delta}1, OSMR{Delta}2, OSMR{Delta}3) resulted in higher expression levels as demonstrated by Western blot analysis (Fig. 5GoB). However, induced heterodimerization of {alpha}/gp130 with truncated ß/OSMR constructs did not lead to increased STAT signals. Indeed, loss of the C-terminal regions of OSMR resulted in less intense EMSA bands (Fig. 5GoC) and reduced {alpha}2-macroglobulin promoter activation in HepG2 cells (data not shown) compared with the combination of full-length receptors. This indicates a function of the OSMR membrane-distal part for signal transduction which is further demonstrated in Fig. 6Go (left panel): a receptor combination devoid of the gp130 STAT recruitment sites ({alpha}/gp130-B1/2 + ß/OSMR) is still able to elicit STAT activation. Apart from the C-terminal region, membrane-proximal sequences of the OSMR also play an important role for signal transduction via an OSMR/gp130 heterodimer: combination of the full-length cytoplasmic part of gp130 with the OSMR box1/2 region resulted in a strong STAT response (Fig. 6Go, right panel). Thus, also the OSMR, as shown for the LIFR above, provides functional motifs necessary for signaling in combination with gp130.



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  		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 {alpha} and ß 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 probe. The bands resulting from STAT3 homodimers are indicated.

 
Jak1 and Jak2 associate with OSMR, LIFR, and gp130

Janus kinases are known to associate with the membrane-proximal region of cytokine receptors. IL-6-type cytokines lead to the activation of Janus kinases Jak1, Jak2, and Tyk2. Therefore, we compared the three receptor subunits gp130, LIFR, and OSMR with respect to their ability to associate with Jaks.

COS-7 cells were cotransfected with expression vectors encoding ß/gp130, ß/LIFR, or ß/OSMR{Delta}1 (we used the deletion construct to achieve higher expression levels, see Fig. 5GoB) and one of the three Janus kinases. After lysis under mild conditions, Jak1 and Jak2 could be coimmunoprecipitated with ß/gp130, ß/LIFR, and ß/OSMR{Delta}1 (Fig. 7Go, upper panels). Binding of Tyk2 could be shown for ß/gp130 and ß/LIFR (Fig. 7Go, lower panel), but not convincingly for ß/OSMR{Delta}1 (data not shown). Jaks did not bind to a gp130 construct with a deletion in the membrane-proximal region including the box1 motif (Fig. 7Go). Moreover, no unspecific precipitation 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-5Rß Ab was used in limiting concentrations (Fig. 7Go).



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  		FIGURE 7. Jak association with gp130, LIFR, and OSMR. COS-7 cells were cotransfected with expression constructs encoding one receptor chimera (ß/gp130, ß/LIFR, ß/OSMR{Delta}1, or ß/gp130{Delta}box1) and one Janus kinase (Jak1, Jak2, or Tyk2), respectively. After lysis of the cells, immunoprecipitates of the receptor and aliquots of cellular lysates were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Western blots were developed with Abs against Jak1, Jak2, Tyk2, or IL-5Rß as indicated. It should be noted that in other experiments the somewhat reduced binding of Jak1 to ß/LIFR (compared with ß/gp130) was not apparent.

 
The signaling capacity of homodimerized LIFR or OSMR cytoplasmic parts depends on the cellular context

Although LIFR and OSMR are only known to participate in heterodimeric receptor complexes with gp130, artificial homodimerization of the respective cytoplasmic regions by chimeric receptors elicited cellular responses in several studies (19, 20, 21, 22, 23, 24) but not in others (24, 25). By combining the respective IL-5R {alpha} and ß chimeras, we studied the effects of homodimerization of the cytoplasmic parts of the LIFR or OSMR. Compared with IL-5R/gp130 homodimers, both the LIFR and the OSMR homodimers yielded only marginal activation of endogenous STAT1 or overexpressed STAT3 in COS-7 cells (Fig. 8GoA). In addition, we replaced the extracellular region of the IL-5R by the one of the EpoR, a homodimer, which has been successfully applied for construction of hybrid receptors (30, 32, 35). However, also the Epo-induced homodimerization of the cytoplasmic parts of LIFR or OSMR elicited only marginal STAT responses, whereas homodimerization of EpoR/gp130 led to a strong signal (Fig. 8GoB). In striking contrast, homodimerized cytoplasmic parts of LIFR and OSMR were able to induce reporter gene activation in HepG2 hepatoma and MCF-7 breast carcinoma cells (Fig. 8GoC). Thus, the signaling ability of LIFR and OSMR homodimers seems to depend on the cellular context.



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  		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 {alpha} and ß or EpoR chimeras as indicated. Right panels, Cells were cotransfected with 5 µg 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 performed using the m67SIE probe. Bands resulting from STAT3 or STAT1 homodimers are indicated. C, Reporter gene assays. HepG2 or MCF-7 cells were transfected with the indicated expression plasmids along with an {alpha}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 ß-galactosidase. The -fold inductions (relative to untreated cells) of three to four independent experiments (mean ± SD) are presented, except for heterodimerization of LIFR and OSMR cytoplasmic parts in HepG2 cells (two experiments).

 
Finally, the IL-5R chimeras enabled us to study the effects of a heterodimerization of the LIFR and OSMR cytoplasmic parts. Even this combination of receptors led to gene activation in hepatoma and in breast carcinoma cells (Fig. 8GoC), underlining the functional homology of these three receptors involved in signaling of all IL-6-type cytokines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we analyzed the contributions of the cytoplasmic parts of LIFR or OSMR to signal transduction in heterodimeric 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 kinases (3, 36). In fact, Janus kinases associate with all three receptors: we could demonstrate binding of Jak1 and Jak2 to OSMR sequences, whereas Jak1, Jak2, and Tyk2 coprecipitated with receptors containing intracellular regions of gp130 and LIFR. It is believed that the presence of (at least) two receptor-associated Jaks is required for initiation of downstream signaling events upon cytokine-induced receptor dimerization (3, 37). Thus, it is very likely that LIFR or OSMR provide the respective "second" Janus kinase 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, 39, 40, 41). Our findings are in accordance with other reports showing that the presence of the membrane-proximal region of LIFR heterodimerized with gp130 is sufficient to mediate gp130 tyrosine phosphorylation, STAT activation, gene induction, or hematopoietic cell proliferation and differentiation (19, 20, 42). However, in contrast to our results, a truncated OSMR retaining the membrane-proximal 60 amino acid residues (comprising box1/2) was found to be unable to support STAT activation and gene induction upon dimerization with gp130 in rat hepatoma cells (19). The usage of different cell types, receptor constructs, and experimental readouts may explain this apparent discrepancy.

Dimerization of the membrane-proximal region of gp130 with the full-length cytoplasmic parts of LIFR or OSMR but not with the respective truncated box1/2 constructs leads to STAT activation (Figs. 4Go and 6Go). This finding demonstrates that 1) the membrane-distal parts of the LIFR or OSMR provide critical STAT recruitment sites, 2) that they are functional even in the absence of the gp130 membrane-distal region, and 3) that only a single chain within such a receptor dimer has to be equipped with STAT recruitment sites. We show for the first time that only the membrane-proximal region of gp130 is sufficient to allow signal transduction when dimerized with full-length LIFR or OSMR cytoplasmic parts. Their ability to activate STATs in the absence of gp130 STAT recruitment sites was so far demonstrated only upon artificially induced homodimerization of the cytoplasmic parts of LIFR or OSMR (19, 20, 21, 22, 23). The contribution of OSMR STAT recruitment modules was also implicated by our finding that successive C-terminal truncations of the OSMR led to decreased STAT responses in COS-7 cells (Fig. 5GoB) and {alpha}2-macroglobulin promoter induction in HepG2 cells (data not shown) when dimerized with full-length cytoplasmic gp130. Intriguingly, however, a C-terminal truncation of only 36 amino acids of the OSMR totally abolished the ability of the OSMR/gp130 complex to activate STATs in Hep3B hepatoma cells (19). In accordance with the study by Kuropatwinski et al. (19), we could demonstrate that C-terminal deletions of the OSMR lead to enhanced protein expression. Future studies will aim at the identification of the molecular basis underlying this phenomenon.

For the chimeric LIFR/gp130 heterodimer, we observed a lower STAT1- and STAT3-activating potential in spite of equal expression levels of the surface receptors (Fig. 3Go). It is very unlikely that this is due to a lower affinity for IL-5 since the affinity of the IL-5R does not seem to be influenced by the receptor’s intracellular region: we have previously demonstrated that the affinity of IL-5R/gp130 chimeras (such as the affinity conversion upon coexpression of a IL-5Rß chimera) is comparable to the one observed for the wild-type IL-5R complex in COS transfectants (26). Also, receptor complexes incompetent of signal transduction such as {alpha}/gp130 + ß/{Delta}cyt (see Figs. 4Go and 6Go) or IL-5R/gp130 chimeras, fused downstream of the transmembrane region, bound IL-5 with normal high affinity (Ref. 26 ; H. M. Hermanns, unpublished data).

Overexpression of STAT3 in COS-7 cells revealed a difference in signaling via OSMR vs LIFR: coexpression of STAT3 significantly increased the STAT signal elicited by heterodimeric gp130/OSMR chimeras relative to that induced by homodimers of the gp130 cytoplasmic part (Fig. 5Go), whereas the signal intensity of heterodimeric gp130/LIFR chimeras remained unchanged (Fig. 3Go). This suggests that the OSMR, compared with the LIFR, may be a more potent activator of STAT3, although it contains only two 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 (YQPQ), Y1001 (YKPQ), and Y1028 (YRPQ).

Cytokines signaling via homomeric LIFR or OSMR complexes (devoid of gp130) are currently unknown. Nonetheless, IL-5 induced homodimerization of the cytoplasmic regions of LIFR and OSMR elicited signals in HepG2 hepatoma and in MCF-7 breast carcinoma cells. In COS-7 cells, however, IL-5R/LIFR or IL-5R/OSMR homodimers, as well as corresponding receptors containing the extracellular part of EpoR, elicited only very weak STAT signals. Several authors have investigated the signaling ability of homodimerized LIFR cytoplasmic chains with partly contradictory results: signaling could be demonstrated in hepatoma and neuroblastoma cells (20), embryonic stem cells (24, 43), and COS-1 cells (21) when chimeric receptors were used that homodimerized upon stimulation with their respective ligands (G-CSF, neurotrophin-3, or epidermal growth factor). On the other hand, G-CSFR/LIFR constructs did not elicit signaling in M1 promyelocytic cells or BAF/03 pre-B cells (24). Similarly, Nakamura et al. (25) did not observe signaling upon GM-CSF-induced homodimerization of two LIFR cytoplasmic parts in embryonic stem cells, although GM-CSF-induced heterodimers of the cytoplasmic parts of LIFR and gp130 elicited a response. However, GM-CSF-induced homodimers of LIFR cytoplasmic regions were able to induce differentiation in M1 and WEHI-3B promyelocytic cells (23). Thus, the cellular background and maybe even the subline used, the transfected hybrid receptors, and the different experimental readouts may affect the results of such studies so that it is difficult to draw general conclusions. It will be of interest to find out to what degree the apparent cell-type specific signaling of chimeric LIFR homodimers might be due to differences in expression of the various Jaks or in their activation profile. The signaling ability of homodimers of the OSMR cytoplasmic region has been addressed so far only in one study (19).

Our study provides evidence that the three signal-transducing polypeptide chains gp130, LIFR, and OSMR relevant for signaling of IL-6-type cytokines have common properties: they critically contribute to signaling via heterodimeric gp130/LIFR or gp130/OSMR receptor complexes and both membrane-proximal and membrane-distal parts are involved. Their functional homology could further be demonstrated by their interchangeability: even enforced heterodimerization of the cytoplasmic parts of LIFR and OSMR elicited reporter gene activation in HepG2 hepatoma and MCF-7 breast carcinoma cells (Fig. 8GoC). Apart from common functions, the differential signaling ability of homodimeric receptors in COS-7 cells points at distinct characteristics of gp130, LIFR, or OSMR, which possibly become evident only in certain cells. Redundant effects of IL-6-type cytokines have been attributed to the shared usage of gp130 as a common signal-transducing chain and the structural as well as functional similarity of gp130, LIFR, and OSMR. Thus, many of the biological effects elicited by a gp130 homodimer can be also observed for the LIFR/gp130 or OSMR/gp130 heterodimer, if the corresponding ligand binding receptor chains are provided. However, several reports have pointed at differences in signal transduction events elicited from gp130/gp130 homodimers and gp130/LIFR or gp130/OSMR heterodimers which cannot be explained by differential receptor expression (e.g., different preferences in STAT activation (19, 44, 45), differentiation of M1 transfectants (17) and PC-12 pheochromocytoma cells (44), and proliferation vs growth inhibition of breast carcinoma cells (46, 47, 48)). Therefore, our receptor chimeras are promising tools to analyze the molecular basis of these differences in signal transduction via heterodimeric gp130/LIFR and gp130/OSMR complexes.


   Acknowledgments
 
We thank Drs. L. Graeve, G. Müller-Newen, F. Schaper, and J. Kuhse for critical reading of this manuscript. We are grateful to Dr. A. Ziemicki (Bern) for providing antiserum against Jak1. We thank Dr. I. M. Kerr (London), and Dr. S. Pellegrini (Paris) for providing the plasmids encoding Janus kinases. Human recombinant Epo was a generous gift from Boehringer Mannheim.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Bonn) and by Fonds der Chemischen Industrie (Frankfurt). Back

2 H.M.H. and S.R. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. I. Behrmann, Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail address: Back

4 Abbreviations used in this paper: OSM, oncostatin M; OSMR, OSM receptor; LIFR, LIF receptor; Epo, erythropoietin; EpoR, Epo receptor. Back

Received for publication May 18, 1999. Accepted for publication October 7, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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