oncostatin m and leukemia inhibitory factor trigger overlapping

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Val. 269, No. 8, Issue of February 25, pp. 6215-6222, 1994 Printed in U.S.A.

Oncostatin M and Leukemia Inhibitory Factor Trigger Overlapping and Different Signals Through Partially Shared Receptor Complexes*

(Received for publication, August 3, 1993, and in revised form, November 9, 1993)

Bettina Thoma$, Timothy A. Bird, Della J. Friend, David P. Gearing$, and Steven K. Dower From the Departments of Biochemistry and §Molecular Biology, Zmmunex Corporation, Seattle, Washington, 98101

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) both bind to the same receptor with high affinity and thus mediate an overlapping spectrum of biological activities, the signal transduction mechanisms for which are unclear. We show that mitogen-activated protein kinases are involved in both the LIF and OSM signal transduction pathways. However, we found that OSM is a much more potent inducer of both mitogen-activated protein kinase activity and biological response, both of which correlate with the expression of a second OSM receptor that does not bind LIF. In addition, different patterns of tyrosine-phosphorylated proteins were stimulated by OSM and LIF. We therefore suggest that the two receptors for OSM can be coupled to different signal transduction events.

Leukemia inhibitory factor (LIF)’ and oncostatin M (OSM), both mediators of pleiotropic biological activities, share many structural and genetic features (1). The two cytokines bind with high affinity to the same receptor (LIF/OSM receptor), which consists of two subunits: LIF receptor Q chain (LIFRa) and gp130 (2). This finding may serve to explain the many overlapping biological responses of both cytokines (for review, see Ref. 3). Several other biological responses seem to be exclusively induced by OSM ( P 7 ) . Because OSM binding can be found in the absence of LIF binding (8), the specificity in signaling could arise from the specificity in receptor binding. The molecular structure of OSM-specific receptors has not been completely elucidated, but has been suggested to consist of gp130 (9), to which OSM binds with low affinity (2), and an unknown protein, which is crucial for high affinity binding and signal transduction, since BAI?BO3 cells transfected with gp130 did not respond to OSM (9). Despite the fact that both receptors for OSM probably include the ubiquitously expressed component gp130 (lo), their relationship and mechanisms of action have so far not been described.

Interaction between many hormone/cytokine receptors and their specific ligands results in activation of MAP (mitogen- activated protein) kinases/extracellular regulated kinases (11). Among these receptors are members of different receptor fami- lies: tyrosine kinases (e.g. epidermal growth factor; 121, immunoglobulin superfamily (e.g. IL-1; 131, hematopoietin receptors

* 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.

$ Postdoctoral fellow sponsored by the Deutsche Forschungsgemein- schaft and Immunex Corporation. To whom correspondence should be addressed. Tel.: 206-587-0430; Fax: 206-233-9733.

The abbreviations used are: LIF, leukemia inhibitory factor; OSM, oncostatin M; M A P , mitogen-activated protein; IL, interleukin; TNF, tumor necrosis factor; PDGF, platelet-derived growth factor.

(e.g. IL-3, IL-5, and granulocyte macrophage-colony-stimulating factor; 14) and the TNF receptor family (e.g. TNF; 15). MAP kinases appear to be intermediates in a signaling cascade (16) from the membrane to the nucleus.

In this study we have compared the mechanisms of LIF and OSM action as a function of their interaction with the LIF/OSM receptor or the OSM-specific receptor. These receptors are differentially expressed, although both may be linked to intracellular events causing activation of MAP kinase. More importantly, our findings demonstrate that OSM induces MAP kinase activity and biological responses more efficiently than LIF and OSM triggers tyrosine phosphorylation of several proteins, which are not stimulated by LIF. These responses correlate with the expression of an OSM-specific receptor. Taken together, the data indicate that different signal transduction mechanisms are elicited by interaction of OSM with its two receptors.

MATERIALS AND METHODS Cell Lines-Most cell lines (HeLa: cervix carcinoma, HepG2: hepato-

cellular carcinoma, JAR: choriocarcinoma, KB: epidermoid carcinoma, SK-HEP-1: liver adenocarcinoma, WI-26 VA4: SV40 transformed lung fibroblasts; all human) were originally obtained from the American Type Culture Collection (Rockville, MD). Cells were either maintained in Dulbecco’s modified Eagle’s medium or RPMI-1640 media supplemented with 10% fetal calf serum (Intergen Co, Purchase, NY), 50 unitdm1 penicillin, 50 pg/ml streptomycin sulfate, and 300 pg/ml L- glutamine. IMTLH-1 cells were obtained by transformation of human bone marrow stromal cells with pSV-neo in our laboratory and were propagated in McCoy’s medium additionally supplemented with 1 mM pyruvate, 5 x M p-mercaptoethanol.

Cytokines, Antibodies, and Reagents-Recombinant human OSM and LIF were produced in yeast and purified as reported earlier (17). LIF used in this paper was produced in a different yeast strain (MNN 9) to reduce the amount of glycosylation and was fully active as determined in a standard proliferation assay using DA1 cells (18). Human interleukin 1 a (IL-1 a) was expressed in Escherichia coli and purified as described previously (19).

A neutralizing rabbit antiserum against human OSM was obtained by repetitive challenge of New Zealand White rabbits with purified yeast recombinant OSM. Anti-phosphotyrosine monoclonal antibody was obtained from UBI (Lake Placid, NY), and anti-human IL-6 serum and monoclonal antibodies were described elsewhere (20). [y-32PlATP (3000 Ci/mmol), NalZ5I, and high molecular weight markers were purchased from Amersham Corp. The iodination reagent Enzymobead was purchased from Bio-Rad, and the enzyme-linked immunosorbent assay developing reagents were from Jackson Laboratories Inc. (West Grove, PA). All other reagents were from Sigma. ST1 substrate peptide, span- ning Thr669 (RRRELVEPLTPSGE) of the epidermal growth factor receptor cytosolic domain, was synthesized and purified as described (13). P81 phosphocellulose discs (2.5-cm diameter) and nitrocellulose mem- branes were obtained from Whatman (Hillsboro, OR).

Radioiodination of Cytokines-Recombinant human OSM and human LIF were radiolabeled with NalZ5I to a specific activity of 2-5 x

described (21). 1015 counts/midmmol using the Enzymobead reagent essentially as

Binding Studies-Adherent cells were harvested with non-enzymatic cell dissociation solution (Sigma). Cells (0.5-2 x 107/ml) were incubated

62 15

62 16 Differential Signal Dansduction by OSM Receptors

with serial dilutions of lZ5I-OSM or lZ5I-LIF, typically beginning with a concentration of 1 x M. Nonspecific binding was determined in the presence of a 200-fold excess of the respective unlabeled ligand. In order to measure type I1 receptors on cells expressing a mixed receptor population, a 200-fold molar excess of unlabeled LIF was included with each concentration of lz5I-OSM. For competition binding studies, the unlabeled molecules were titrated serially, starting at a concentration of 1 x

M, and the lZ5I-labeled cytokines were added a t a constant concentration of 3 x 10-lo M prior to addition of cells. Binding reactions were incubated for 2 h a t room temperature. Bound lZ5I-ligand was separated from unbound ligand on phthalate oil columns as described (22). Scatchard analysis of binding curves was carried out using the program RS/1 (Bolt, Beranek, and Newman, Boston, MA).

Stimulation of Cells and Preparation of Extracts-Cells were seeded into 6-well tissue culture dishes (Costar, Cambridge, MA) a t a density of 0.25-1 x 106/well, depending on the cell type used, to give a confluent monolayer. After 24 h of incubation, cells were pretreated for 3-5 h at 37 "C with 1 ml of culture medium containing 1% bovine serum albumin to starve cells from fetal calf serum-contained growth factors. Cells were stimulated by addition of 10 pl of appropriate dilutions of cytokines or antibodies in culture media, 1% bovine serum albumin, and incubated at 37 "C for either various lengths of time (time course experiments) or 10-15 min, depending on the cell type used, in order to achieve maximum stimulation of MAP kinase (i .e. dose-response experiments). Thereafter, monolayers were extracted as described previously (13). Briefly, cells were scraped into 400 pl of ice-cold extraction buffer (20 mM HEPES, pH 7.4.30 mhtp-nitrophenyl phosphate, 10 mM NaF, 10 mM MgClZ, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM Na3V04, 0.1 mM Na,Mo04, 10 mM sodium 6-glycerophosphate, 1 mM phenylmethylsul- fonyl fluoride, 10 PM leupeptin, 10 p~ pepstatin A) and disrupted by repeated passages through a 25-gauge needle. Extracts were cleared of cellular debris by centrifugation for 30 min at 10,000 x g and either assayed immediately or stored a t -70 "C.

Measurement of MAP Kinase Actiuity-Assays were performed essentially as described (13). Briefly, 10 pl of extract was added to 20 pl of kinase reaction mixture containing kinase substrate (0.75 mM ST-1 peptide) or water for determination of peptide or nonspecific phosphorylation, respectively. Reactions were carried out for 20-25 min at 30 "C and terminated by addition of formic acid. After removing insoluble material by a brief centrifugation step (IO min, 10,000 x g), 30 pl of the reaction mixture was spotted onto P81 phosphocellulose filters. Filters were washed, dried, and Cerencov-counted.

Measurement of IL-6 Protein Production-KB cells (1 x 104/well) were seeded into 96-well flat bottom microtiter plates. After cells had formed an adherent monolayer, they were stimulated in duplicate with OSM or LIF for 48 h a t 37 "C, and IL-6 in the supernatants was measured by enzyme-linked immunosorbent assay, essentially as described (20). Briefly, conditioned media were incubated in anti IL-6 monoclonal antibody-coated microtiter plates. Plates were then developed by an incubation with a rabbit polyclonal IL-6 serum followed by horseradish peroxidase-coupled goat anti-rabbit immunoglobulin. Immune-complexes were quantified using 3,3',5',-tetramethyl benzidineiH202.

Growth Inhibition Assay-2-5 x lo3 cells were seeded into 96-well flat bottom microtiter plates and allowed to adhere for 12 h. Monolayers were then treated with serial dilutions of OSM or LIF for 4 days at 37 "C before they were stained with crystal violet and plates were measured a t 562 nm as described (23).

Anti-phosphotyrosine Western Blotting-Cytosolic extracts (preparation see above) were diluted with SDS sample buffer, resolved on 8-16% gradient SDS gels, and subsequently transferred onto nitrocellulose. After blocking with 10% bovine serum albuminPBS, the membrane was developed by incubation with anti-phosphotyrosine monoclonal antibody followed by an anti-mouse Ig-biotidavidin-alkaline phosphatase detection system. Nonspecific binding of anti-phosphotyrosine antibody was determined in the presence of 5 m o-phospho-L-tyrosine.

RESULTS

Different Human Cell Lines Express Different Populations of LIF 1 OSM Receptors--Two significant observations have been made recently regarding the nature of OSM receptors. First, OSM binds in a cross-competitive manner to the LIF/OSM receptor (2), and second, some cells have been reported to bind OSM but not LIF (OSM-specific receptor; 8). For clarity in what follows, we shall refer to the receptor which binds both OSM and LIF and which appears to contain the LIFRa and gp130 (2) as the type I receptor. The type I1 receptor binds only OSM.

0 e 0 Labelled ligand OS M LIF OSM LIF

competitor OSM OSM LIF LIF

I O o o n JAR

if 20

1w

80 1 f

20 40"-

IO" I O ' O I O9 I o n IO'

Concentration (Molar)

lines. Binding of a constant amount (3 x 10"O M) of either lZ5I-OSM FIG. 1. Inhibition of 12sI-OSM or lZSI-LIF binding on human cell

(open symbols) or Iz5I-LIF (closed symbols) was determined in the presence of serial dilutions of unlabeled OSM (0) or LIF (0) on three different human cells (HeLa, HepG2, and JAR) as described under "Ma- terials and Methods." 100% inhibition was set as the level of binding observed in the presence of 2 x M homologous unlabeled ligand. The

version to I (%) uersus concentration (M), by non-linear fitting of a data were analyzed, after subtraction of nonspecific binding and con-

one-site competitive inhibition model (parameters I , (%) and K, (",)). In those cases where data were clearly more complex as judged by the

two-site model was used (parameters I , (%I, K, (M-,) and I, (%), K2 difference between experimental and theoretical values for I (%I, a

(M-,)). In no case was a model more complex than the non-cooperative two-site version required to account for the data. Parameter values for the individual cell lines were as follows. HeLa: 0, K , = 3 1 x IO'O, I , = 73 14, Kz = 3 3 X IO', I2 = 30 2 12; 0, Kl = 9 2 4 X lo'', I1 = 31 * 4, Kz = 5 f 8 X lo7, I , = 20 2 8; W, K, = 1 2 0.2 x lo'', I , = 70 f 4, K2 = 3 f 4 x lo7, I , = 27 f 13; 0, K, = 5.7 2 0.9 x lo9, I , = 95 f 4. HepG2: 0, K, = >1013, I , = 28 5, K, = 2.3 2 0.7 x 109, z2 = 75 5; 0, K, = 1.7

X 108, I , = 92 IO; 0, K, = 2.0 2 0.2 X 109, I , = 97 2; W, K, = 9 2 2 X

0.6 X lo'', I1 = 41 f 2; W, Kl = 3 f 7 X lo'', I , = 82 2 3; 0, K, = 3.6 f 1.0 x lo9, I , = 85 f 5. JAR: 0, K, = 1 f 7 x loll, I , = 11 f 11, Kz = 3.2 f 1.0

IO9, I , = 45 f 10, K, = 3.0 t 0.7 x lo*, I , = 56 2 10; 0, K, = 5 f 12 x lo", I l = 17 f 6, Kz = 1 f 4 X IO', I , = 58 2 6 .

Fig. 1 illustrates the distribution of these two types of receptors on three cell lines, JAR, HeLa, and HepG2, as determined by competing unlabeled OSM or LIF against a fixed concentration of lZ5I-OSM or lZ5I-LIF, respectively. On JAR cells, the data show that all lZ5I-OSM binding could be blocked by high concentrations of unlabeled LIF and that >70% of lZ5I-LIF binding could be blocked by high concentrations of unlabeled OSM. The residual -30% lZ5I-LIF binding is interpreted as excess expression of LIFRa, which binds LIF with low affinity (IC, 5 x M, Table I), but does not bind OSM (2). Thus most of the receptors on JAR cells appear to be type I receptors. There are some subtleties in the binding properties of the receptors, observed as a larger shift in the inhibition curves for OSM versus lZ5I-LIF relative to that of OSM versus 1251-OSM than is the case for the corresponding pair of unlabeled LIF curves. However, the differences are small. The overall affinity of OSM for the type I receptors on JAR cells is 5-10-fold lower than that of LIF. These data closely resemble those previously described for cells transfected with various combinations of LIFRa and gp130 (2). By contrast, HepG2 and HeLa cell lines

Differential Signal Dansduction by OSM Receptors TABLE I

Binding of 1251-OSM and Iz5I-LIF to various human cell lines

6217

Cell line L" L C b KG 1 Sitedcell K-2 sitedcell No.'

HeLa

HepG2

KB

WI-26 VA4

SK-HEP-1

IMTLH

JAR

-

1251-labeled ligand.

OSM OSM LIF LIF

OSM OSM LIF LIF

OSM OSM LIF LIF

OSM OSM LIF

OSM OSM LIF

OSM LIF

OSM OSM LIF LIF

LIF

OSM

LIF

OSM

LIF

OSM

LIF

LIF

LIF

OSM

"1

6 3 x 1O'O

3 f 4 x 10'2 5 f 4 x 10'0

1.5 f 0.2 x 10"

3.1 f 5 x 10'O 1.5 2 1 x 10'O

3 2 1 x 10'0 2 f 2 x 10'0

3 f 3 x 10'0 6 3 x 10" 1 -c 0.6 x 10"

ND

>lo" >lo"

1 f 1 x 10'2

6.5 f 1 X 109 8 1 x lo9

ND

2.5 f 2.2 X 109 ND

1.1 f 0.1 x 10'0 8 7 x 1 O ' O

ND 9 f 7 x 10'0

(Range ) 0-600 -c 300 8-300 300 0-900 2 500 40-90 f 10

120-550 -c 150 100-600 2 300 80-500 2 60 0-80 ? 10

20-200 f 100 5-20 * 12

6 1 0 0 f 100 0

0-70 f 10 0-90 f 20

70-130 f 10

400-700 f 30 600-700 -c 200

0

3,10041,000 -c 1,000 0

600-1,300 f 200 40-70 f 30

0 800 2 400

1.3 2 0.5 x lo9 "1

1.5 f 0.7 x lo9 4.5 -c 1.7 x lo8 2.5 f 1.5 2 lo9

1.2 -c 0.9 x 109 3.5 f 2 x 108

NDd ND

1.5 f 0.3 x lo9 2.8 & 0.7 x lo9

ND 3 -c 0.5 X 109

1.7 f 0.5 x lo9 1.4 f 0.5 x lo9

ND

2 f 0.3 x lo8 2 -c 0.3 x lo8

ND

ND ND

3 x 108 ND

2.3 f 0.7 x lo9 5 f 2 X 109

2,900-21,000 * 7,000 (Range )

1,100-11,000 2 3,500 1,200-8,lOO f 2,000 9004,100 f 1,000

1,50%3,100 f 600 1,500-12,000 f 5,000

0 0

800-3,000 f 2,000 180-1,100 f 100

25-820 f 110 0

19004,800 f 700 16004,850 2 700

0

0-6,300 2 800 04,000

0

0 0

3,000-10,000 0

6,000-7,000 f 2,000 1,300 f 200

4 4 3 3

5 5 5 3

3 3 3 1

3 3 2

2 2 2

4 1

2 2 3 2

Unlabeled ligand in 200-fold excess to '251-labeled ligand. Number of observations. Not detected.

displayed a mixed population of type I and I1 receptors as determined by cross-competition studies. Essentially all the lZ5I-LIF binding could be blocked by high concentrations of OSM, yet only 50% of the lZ5I-OSM binding could be blocked by LIF, even at LIF concentrations -100-fold higher than those capable of blocking all Iz5I-OSM binding to JAR cells. These data suggest that at the concentration of 1251-OSM used in the experiments (10"O M), approximately 50% was bound to type I receptors and 50% to type I1 receptors. The type I1 receptor cannot be simply free gp130, shown to bind OSM with low affinity (K, = los M - ~ ; 2), since all lZ5I-OSM binding to HeLa or HepG2 cells could be be blocked by unlabeled OSM with an inhibition constant >lo9 M-I. It follows that the labeled ligand was associated with a receptor that binds OSM with an affinity at least 10-fold higher than does gp130.

In order to examine the binding properties of the receptors in more detail, we performed a series of titrations of labeled OSM or LIF with several different cell lines. For those cells where significant binding of both labeled cytokines was detected, we attempted to distinguish shared (type I) from specific (type 11) receptors by examining binding of labeled LIF t unlabeled OSM and labeled OSM f unlabeled LIF, arguing that in the presence of a large molar excess of competitor only binding of labeled OSM to specific (type 11) receptors would be detected. The data from representative experiments plotted in the Scatchard coordinate system are summarized in Fig. 2. Binding site numbers and affinities, determined by non-linear least squares curve fitting of non-cooperative one or two site models to a more extensive data set, are summarized in Table I.

Two of the cell lines examined (IMTLH and SK-HEP-1) showed no detectable Iz5I-LIF binding and a single class of 12'I-OSM binding sites with an affinity >lo9 M - ~ . We examined '251-OSM binding to SK-HEP-1 in the presence of excess unlabeled LIF to confirm the specificity of these type I1 receptors.

No significant changes in 1251-OSM binding were observed (Fig. 2). A third cell line, WI-26 VA4 had a very low level of LIFIOSM receptors, ranging from 1-10% of the level of total OSM receptors. This number corresponded to the number of high affinity OSM-binding sites, and these sites are most likely type I receptors. As expected from the low level of LIF binding, no significant effect of excess unlabeled LIF on lZ5I-OSM binding was observed (Fig. 2). The affinity of lZ5I-OSM binding indicates that the type I1 receptor on these cells is distinct from gp130, although the receptor may be a multichain structure of which gp130 is part (9).

The binding data for the other four cell lines (HepG2, KB, HeLa, and JAR) were more complex. For the simplest case, JAR cells, it is clear that the direct binding experiments revealed some complexity not apparent from the data in Fig. 1. The majority of the receptors appeared to be of the cross-reactive type I class. Nevertheless, there was residual binding of lZ5I- LIF in the presence of excess OSM and to some extent vice versa. Most importantly, however, there appeared to be virtu- ally no type I1 OSM receptors (Fig. 2 and Table I). The residual low affinity Iz5I-LIF-binding can be explained by excess expression of the LIFRa. The very low numbers of residual high affinity LIF-binding sites in the presence of excess OSM are likely due to incomplete competition of labeled ligand binding to the type I receptor, since a higher molar excess of unlabeled OSM will block completely (data not shown).

Three other cell lines (HepG2, KB, and HeLa) had binding properties that appeared to arise from a mixed population of type I and I1 receptors. HepG2 cells displayed a small number of relatively high affinity 1251-LIF-binding sites, all of which could be blocked by excess OSM, and a much larger number of lZ5I-OSM-binding sites with intermediate to low affinity. Addi- tion of excess LIF only eliminated a fraction of the highest affinity OSM-binding sites. These data can be accounted for by

62 18

3.0

2.0

1 .O

0 - h

'0 0.8 X e, 0.6 5 0.4

v

E 0.2

m 0.8

06

0 4

0 2

Differential Signal Dansduction by OSM Receptors

0 a 0 B Labelled ligand OSM OSM LIF LIF competitor LIF OSM

SK-HEP-I

1.0 2.0 3.0 4.0 2.0 4.0 6 0 8.0

Molecules Bound/cell (x IO3 ) FIG. 2. Comparison of 12SI-OSM and lZ6I-LIF binding on differ-

ent human cell lines. Scatchard analysis of lZ5I-OSM binding in the absence (0) or presence of unlabeled LIF (0) and of lZ5I-LIF binding in the absence (0) or in the presence of unlabeled OSM (W) are shown. Association constants (K, ( M - ~ ) ) and siteskell (r) were determined by non-linear least-squares fitting to the data. Where K, values were too low to allow independent estimation of r and K,, but a low affinity site was clearly present, binding to that site is summarized as the product K, . r (molecules cell-' M-,). Values for each individual curve were as follows. HeLa: 0, Kal = 9 x lolo, rl = 270, KO, = 1.2 x lo9, r, = 11,400; 0, K,, = 9 x lo", r l = 52, K,, = 1.6 x lo9, r2 = 3,950; 0, K,,, = 1 x lo", r l = 130, K,, = 3.7 x lo9, r2 = 5,250; W, KO, = 1.8 x 10". rl = 36, K,, = 7.1 x lo8, r2 = 861. HepG2: 0, K,, = 1.3 x lolo, rl = 540, Kazrz = 1.3 x

= 550; K,, = 3.3 x lolo, rl = 76. WI-26 VA4: 0, K,, = 1.6 x lo9, rl = 10l2; 0, K,, = 6 x lo9, rl = 380, Ko2r2 = 8 x 10"; 0, K,, = 1.5 x lolo, rl

4,800; 0, KO, = 1.8 x lo9, rl = 4,850; 0, K,, = 2.5 x lolo, rl = 130. IMTLH: 0, K,, = 2 x lo9, rl = 12,500; 0, not detected. SK-HEP-1: 0, KO, = 9 x lo9, rl = 680; 0, K,, = 7 x lo9, rl = 660. JAR: 0, K,, = 1 x lO 'O, rl = 620, Ka2r2 = 3 x lo',; 0, KO, = 7 x lo9, r l = 40; 0, KO, = 4 x lo9, rl = 6,400, K,,r, = 1 x 1013; W, K,, = 2 x lolo, rl = 750, Ka2r2 = 4 x 10l2.

a model in which these cells express gp130, a putative OSM receptor and the LIFRa, the last at limiting levels. The data for KB cells (Table I) resembled those for the HepG2 cells.

HeLa cells showed the most complex behavior, having receptors with high and low affinity for lZ5I-LIF and 1251-OSM and similar numbers of total receptors for both cytokines. In agree- ment with the competition data in Fig. 1, excess LIF reduced lZ5I-OSM binding by about 50% and appeared to do so by blocking 50% of the OSM-binding sites rather than affecting the affinity of either component of the 1251-OSM-binding curve (Fig. 2). All lZ5I-LIF binding was competed by excess OSM in cross- competition experiments (Fig. 1). However, data from binding studies with lZ5I-LIF in the presence of unlabeled OSM (Table I) suggested that there is probably LIFRa expressed on HeLa cells not detected in the competition experiments at the low concentration of lZ5I-LIF used. As seen with JAR cells, a higher molar excess of unlabeled OSM competed the residual high affinity lZ5I-LIF binding (data not shown), which might in both cases be due to the lower affinity of the LIFIOSM receptor for OSM than for LIF (Fig. 1; 2).

In summary, the JAR cells express mostly type I receptors but more importantly no type I1 receptors. The SK-HEP-1 and IMTLH cells express exclusively type I1 receptors. The WI-26 VA4, HepG2, and KB lines express primarily type I1 receptors and type I receptors at 1-30% the level of type 11, with WI-26 VA4 cells being at the low end and HepG2 cells at the high end

l4 t HeIa t IMTLH

c 2 6l

Time (Minutes) FIG. 3. Transient activation of MAP kinase by OSM and LIF in

various human cell lines. Confluent monolayers of cells were stimulated with OSM (0) or LIF (0) for various lengths of time at 37 "C. Cytosol extracts were prepared immediately and assayed in triplicate incubations for MAP kinase activity ("Materials and Methods"). Values for M A P kinase activity are expressed as "stimulated-unstimulated" phosphate incorporation (10-l pmoVmi1d2.5 x lo4 cells). Standard errors calculated were smaller than size of symbols and were therefore not included. Shown are representative experiments of a least three rep- etitions.

of this range. Finally, HeLa cells showed more complex binding isotherms, but the data can likely be explained if type I, type I1 receptors, and some free LIFRa are present on these cells. The binding data obtained for HeLa cells cannot be completely accounted for by a simple two-chain model of the type proposed by Gearing and co-workers (21, since binding sites with high and low affinity for Iz5I-LIF and 1251-OSM were reduced in presence of cold competitor. The subunit composition of the receptors may be more complex; however, tools (e.g. blocking receptor antibodies) are not yet available to address those questions.

Activation of MAP Kinase-In most human cell lines examined, OSM induced a marked and transient increase in MAP kinase activity, detectable after 5 min, reaching peak levels around 10-12 min post stimulation and subsequently declining to basal levels after about 60 min (Fig. 3). In only one cell type (HeLa) was LIF capable of stimulating a pronounced activation of M A P kinase with similar kinetics, albeit to a significantly lower maximal level compared to OSM (Fig. 3). All other cell types responded to LIF with insignificant (HepG2, WI-26 VA4, SK-HEP-1, IMTLH) or very low (KB, JAR) activation of MAP kinase (Table 11, Fig. 3). OSM was the superior inducer of kinase activity in these cell lines except for JAR cells. Its activity was not due to some trace contaminant, since it could be blocked efficiently by a neutralizing rabbit anti-OSM serum (data not shown). In JAR cells, the level of OSM-stimulated MAP kinase activity was the lowest of all cell types tested and, unusually, equalled the level of the LIF response. Comparing the response pattern of OSM and LIF in all cell lines with the expression of cell surface receptors for both ligands (Tables I and 11), we observed that efficient stimulation of MAP kinase by OSM correlated with expression of type I1 OSM receptors. First, in all cell lines expressing type I1 receptors, MAP kinase was induced by OSM; second, not all cells expressing the type I receptor showed M A P kinase activation in response to LIF, and third, OSM and LIF were similarly weak in stimulating MAP kinase activity in a cell line (JAR) bearing type I but not

Differential Signal Dansduction by OSM Receptors TABLE I1

Activation of MAP kinase in various human cell lines

6219

Cells were stimulated with saturating concentrations of cytokines (5 X lo-' M OSM, LIF, EGF; 1 X lo-' IL-1).

Cell line Q p e of OSM R Agonist stimulated MAP kinase activity" expressed OSM LIF IL-1 EGF

HeLa I + I1 9.860 ? 2.227 4.206 ? 1.226 ND 8.010 n = 17 n = 17 n = l

HepG2 I + I1 5.010 ? 1.572 0.741 ? 0.895 8.593 f 3.418 ND n = 17

KB n = 17 n = 4

n = 6 WI-26 VA4 I + I1 13.35 t 2.997 0.551 ? 0.313 9.310 2 1.602 ND

n = 5 n = 3

n = 3 n = 3 n = 2

IMTLH I1 7.066 f 0.634 ND n = 8

6.872 f 1.588 n = 7

0.037 ? 0.06

JAR I 2.453 ? 0.839 n = 3 n = 4

2.764 t 1.009 9.856 f 4.116 n = 2

ND n = 10 n = 10 n = 4

I + I1 4.320 ? 0.821 1.040 f 0.263 3.896 ? 0.862 ND

SK-HEP-1 I1 3.292 2 0.783 0.406 * 0.34 ND 2.874 f 0.498 n = 2

a Values are given as mean ? standard error of phosphate incorporation (10-l pmoVmid2.5 x lo4 cells). Not determined.

type I1 receptors. We examined whether signaling via the type I receptor trig-

gered MAP kinase activity less efficiently than signaling via the type I1 receptor, or whether OSM and LIF signaled differ- ently through the type I receptor as do, for example, IL-1 and IL-1 receptor antagonist through the p80 IL-1 receptor (24). In HepG2 cells, which express both types of OSM receptors, MAP kinase activation by OSM (1 x M) was not inhibited by the presence of a 100-fold excess of LIF (1 x M; Fig. 4). Using similar molar ratios of OSM and LIF in a binding competition experiment, 1251-OSM binding to the type I receptor was completely inhibited by LIF, and the remaining non-competable binding was due to OSM binding to the type I1 receptor (Fig. 1). Therefore, MAF' kinase activation in HepG2 cells was induced largely via binding of OSM to the type I1 receptor. This view is supported by the similarly low levels of MAP kinase induction by both cytokines in JAR cells, which do not express type I1 receptors.

Fig. 5 shows dose-dependent induction of MAP kinase activity by OSM and LIF in several cell lines. Half-maximum stimulation of MAP kinase was achieved at a concentration of 2-6 x 10-lo M, which was in the range of the dissociation constants of the two cytokines (Table I). At saturating concentrations (0.5-1 x M), the amount of MAF' kinase activated by OSM varied between the different cell types examined. Highest levels were induced in WI-26 VA4 and lowest levels in JAR cells (Figs. 3 and 5, Table 11). The differences in kinase activation did not correlate with numbers of OSM receptors expressed on the cells. For example, IMTLH cells express 10-fold more binding sites when compared with WI-26 VA4 cells, yet 2-fold higher levels of stimulated MAP kinase activity were found in WI-26 VA4 than in IMTLH (Tables I and 11).

In all cell lines expressing the type I1 receptor, OSM was an equal or better inducer of MAP kinase activity than epidermal growth factor or IL-1 (Table 11), both known to be potent acti- vators of MAP kinases (12,131. By contrast, IL-1 stimulation of MAP kinase in JAR cells far exceeded the level of MAP kinase activation by OSM or LIF, suggesting that JAR cells contain a pool of MAP kinase inducible by external stimuli which is re- fractory to OSM or LIF stimulation (Table 11). This suggests that in these cells the limiting factor was neither the available pool of enzyme or some other generic component in the MAP kinase pathway. Taken together, these data indicate that OSM interaction with the type I1 receptor is capable of strongly ac- tivating MAP kinase, whereas interaction of either OSM or LIF with the type I receptor generates a much weaker signal.

Expression of OSM Qpe 11 Receptors Correlates with Biologi-

T

OSM - + + LIF - + - +

FIG. 4.OSM-stimulated MAP kinase activity is not inhibited by LIF. Confluent monolayers of HepG2 cells were either left untreated, stimulated with OSM (lo-' M) or LIF M), or both together as indicated by - or +. Cells were incubated at 37 "C for 12 min. Cytosol extracts were assayed for MAP kinase activity in triplicate incubations as described ("Materials and Methods"). MAP kinase activity is expressed as total phosphate incorporation (10-l pmoles/mid2.5 x lo4 cells). Values of five independent experiments are shown as mean f S.E.

cal Responses-We analyzed biological responses to OSM and LIF, IL-6 production, and growth inhibition (1, 5) to assess whether there exists a correlation between expression of receptors and/or activation of MAP kinase. OSM stimulated KB cells to secrete IL-6 and inhibited proliferation of WI-26 VA4 or HeLa cells in a dose-dependent manner (EDSO 1-4 x 10-lo M;

Fig. 6). LIF was a much weaker inducer of IL-6 production in KB cells, correlating with the weaker activation of MAP kinase found in this cell line (Table 11). In addition, an inhibitor of MAP kinase activity (quercetin; 20) was capable of greatly reducing the level of IL-6 produced (data not shown), implicating MAP kinase in IL-6 production stimulated by OSM or LIF, as previously shown for IL-1(20). However, within the time frame of MAP kinase activation, the cells do not appear to commit completely to a biological response, as the onset of IL-6 secre- tion was found to be much slower than peak activity of MAP kinase (Fig. 3 and data not shown; 20). Thus, it seems conceiv- able that additional signals besides MAP kinase activation are required. Inhibition of proliferation could be correlated to the expression of type I1 receptors but not to activation of MAP kinase, as in HeLa cells LIF was not capable of inducing a growth inhibitory response.

6220 Differential Signal Dansduction by OSM Receptors

l o ' ' > lo'"' I O " 1 o 1 2 lo'"' 1 o n

Concentration (Molar) FIG. 5. Dose-dependent activation of MAP kinase. Cell monolay-

ers were stimulated with serial dilutions of either OSM (0) or LIF (El) ranging from 10"2--10" M. After incubation a t 37 "C for 10-15 min, dependent on the cell line used, kinase extracts were prepared and MAP kinase activity was measured in triplicate incubations as described under "Materials and Methods." Standard errors calculated for each value did not exceed the size of the symbol. A representative of several independent experiments is shown for each cell line tested. Values for MAP kinase activity are plotted as stimulated-unstimulated phosphate incorporation (IO" pmoles/min/2.5 x IO' cells).

I

0.6

0.4

0.2

0.0 I& . . . . . . . . . . . . . . . . , , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .

V I A I I

0 IO'O lo" Agonist ConcenIntion (Molar)

FIG. 6. OSM is a more effective inducer of biological responses in human cells. Upper panel, KB cells were cultured for 48 h in presence of serial dilutions of OSM (0) or LIF (0). Supernatants were harvested and assayed for IL-6 in an enzyme-linked immunosorbent

panel, WI-26 VA4 (open symbols) or HeLa cells (closed symbols) were assay. Data shown represent mean values of triplicates ? S.E. Lower

incubated for 4 days with serial dilutions of OSM (0) or LIF (El). There- after, the growth inhibitory effect of OSM or LIF was determined by staining the cells with crystal violet. Mean values of six replicates (SD < 15%) are shown as AOD,,, nm (untreated control-treated sample).

OSM and LIF Stimulate prosine Phosphorylation of Several Cytosolic Proteins--Tyrosine phosphorylation is necessary for the enzyme activity of I" kinases (25). As shown in Fig. 7, OSM or LIF induce tyrosine phosphorylation of proteins with a molecular mass of 42 and/or 44 kDa, presumably one of the two well known species of the MAP kinase family (11). The amount of tyrosine-phosphorylated MAP kinase detected by Western blotting correlates with MAP kinase activity induced by both ligands in each individual cell line. As only 1 tyrosine residue/ active kinase molecule is phosphorylated (25), we conclude that

Cell Line HepG2 HeLa

200-

ag - i d J -200

45- - . -45 " .

FIG. 7. Analysis of tyrosine phosphorylation stimulated by OSM or LIF in human cells. Cytosol extracts of untreated cells or cells treated with OSM or LIF (12 min, 37 "C) were prepared as described and resolved under reducing conditions by SDS-PAGE. Sepa- rated proteins were transferred onto nitrocellulose and subsequently probed with an anti-phosphotyrosine antibody.

signals transmitted by the type I1 receptor recruit more MAP kinase molecules into an active state.

In addition, several other tyrosine-phosphorylated proteins were detected. Both LIF and OSM induced tyrosine phosphorylation of a -110-kDa protein in HeLa cells and a -70-kDa protein in HeLa and HepG2 cells. Interestingly, only upon stimulation with OSM additional tyrosine-phosphorylated proteins were detected: -90 kDa in HepG2 cells and -130, 150, and 190 kDa in HeLa cells. The differences in the tyrosine phosphorylation pattern induced by OSM or LIF more readily suggest that the two receptors for OSM elicit distinct signal transduction events. It is not clear yet whether there exists a causal relationship between any of the tyrosine phosphorylated proteins and MAP kinase activation.

DISCUSSION

OSM and LIF are genetically and structurally related poly- functional ligands, capable of utilizing several receptors (LIF/ OSM receptor or type I, OSM-specific receptor or type 11). Other examples of such systems are the isoforms of IL-1, TNF, or PDGF and their receptors, which are expressed differentially (23,26,27). We show here that two types of OSM receptors can be expressed independently on human cell lines. LIF and OSM receptors belong to a class of receptors characterized by multiple ligand-binding chains interacting with a common subunit to yield a high affinity receptor complex capable of transducing signals. The properties of such receptors are usually discussed within the framework of a two-chain model (2,3,28). However, the binding data obtained in this study are too complex to be entirely accounted for by such a model. More complex struc- tures have been suggested by recent data for heterotrimeric IL-2 and ciliary neurotrophic factor receptors (29,30), homodi- meric growth hormone receptors complexed with a -120 subunit (31), and homotrimeric TNF receptors (32). It has also been shown that gp130 can dimerize (33). If in the LIF/OSM receptor system homo- or heterodimers and/or -trimers can be formed, then when three individual receptor components (e.g. LIFRa, OSMRx, and gp130) are coexpressed, as many as 16 different receptor complexes could be present. This model may explain the broad range of dissociation constants for LIF and OSM binding observed in this paper and others (8,34). A more complex structure of the LIF receptor involving a third subunit was discussed (2) because transfection of COS-7 cells with the LIFRa and gp130 resulted in intermediate rather than high

Differential Signal TFansduction by OSM Receptors 6221

affinity LIF-binding. In addition, Linsley and colleagues (35) suggested a minimum of two OSM receptor components to explain the binding isotherms for OSM. Based on the results obtained from seven human cell lines in this report, there seems to be no correlation between responsiveness to LIF (induction of MAP kinase in HeLa cells uersus KB or HepG2 cells) and LIF binding. The IL-2 receptor system can be viewed as a similar case: high affinity IL-2 binding was generated by co- transfection of the IL-2 receptor a- and p-chain, yet lack of responsiveness in fibroblasts led to the cloning of the y-chain, which is essential for signaling (29). Although there are numer- ous questions to be answered concerning the components of the LIF and OSM receptor system, it seems clear from the data provided in this report and by others that some of these components form a non-cross-reactive receptor entity, the OSM- specific receptor.

Our data suggest that the two receptors for OSM may be functionally different, since they are capable of transducing qualitatively (tyrosine phosphorylation) as well as quantita- tively ( M A P kinase induction, growth inhibition, and IL-6 production) different signaling events within the same cell line. In addition, stronger induction of acute-phase proteins by OSM than LIF was observed in HepG2 cells (6). The reasons for coexpression of multiple receptors with parallel and overlapping functions are not yet fully understood. In the IL-1 system, it appears that only the type I receptor mediates signaling, whereas the type I1 receptor represents a membrane-bound precursor for a soluble IL-1 receptor (36). Although soluble LIF receptors have been found (17, 371, only membrane-bound LIF receptors have been shown to be capable of transducing signals (38). In the TNF receptor system, functional similarities or differences between the two TNF receptors have only been assessed at the level of biological responses (23,39,40). PDGF receptors are a well studied receptorfiigand system in which receptor structure, diversity of signals, and biological function have been compared. Eriksson and colleagues (41) have shown that some intracellular molecules were commonly associated with both receptor isoforms whereas others were only bound by the PDGF-p receptor, which was capable of stimulating chemo- taxis. However, the signal diversity observed at the receptor level has not been linked MAP kinase, known to be activated by PDGF (42).

The findings reported here for the two OSM receptors provide evidence that an intermediate step of a signaling cascade, i.e. MAP kinase, can reflect receptor diversity. In contrast to PDGF receptors, not all components of the type I1 OSM receptor are characterized, and it will be interesting to see whether our observations can be connected to more receptor proximal events. p21"" (431, MEK kinase (44), and Raf-1 kinase (45) are molecules possibly involved in MAP kinase activation. How this relates to the finding that OSM activates pp6P'" (46) will be a matter of further analysis.

While the putative ligand-binding chains of the type I1 OSM receptor have yet to be characterized at the molecular level, it seems clear that gp130 is involved in both ligand binding to and signaling through this receptor, although it cannot transduce OSM signal on its own (9). It remains to be determined whether a 160-kDa OSM receptor protein characterized by cross-linking (35) is identical to gp130 or is a different subunit capable of generating a functional receptor complex. In the case of IL-6, a composite receptor structure including gp130 is essential for signal transduction (10). Gp130 is needed for the formation of the LIF/OSM receptor and presumably accounts for the redun- dancy in LIF and IL-6 responses in many cases (3). However, the LIFRa seems to be able to modify a "gp130 signal." For example, LIF and IL-6 differ in induction of acute-phase genes (47) and the cytoplasmic domain of the LIFRa appears to con-

tribute to LIF/OSM signaling in hepatoma cells (38). Non-overlapping activities of OSM and LIF (3) might be explained by the findings described herein. However, if the type I1 OSM receptor forms a complex with gp130 it is possible that an unknown ligand binding chain contributes to or modifies the signal out- put of such a complex. On this basis, an overall unifying function of gp130 within a "gp130 receptor family" becomes ques- tionable. Individual ligand-binding chains would then provide more than ligand binding function, in contrast to the current view of the IL-3, IL-5, and granulocyte macrophage-colony- stimulating factor receptor family (14). In the case of the OSM specific (type 11) receptor, the undefined a-chain is of great interest, as OSM but not LIF promotes growth of Kaposi's sarcoma cell (7) and therefore the protein itself or associated signal transduction molecules may be prime targets for OSM antagonists.

Acknowledgments-We are grateful to J . McGourty, S. Novick, J . A. King, and V. Price for production of hu LIF and human OSM, to N. Boiani for providing rabbit serum against human OSM, and to John Sims, Bruce Mosley, Anne Bannister, and Steve Gillis for reviewing the manuscript.

1.

2.

3. 4.

5.

6 .

7.

8. 9.

10.

11.

12.

13.

14.

15.

16. 17.

18.

19.

20.

21.

23. 22.

2'4.

25.

26.

27. 28.

29.

30.

REFERENCES Rose, T. M., and Bruce, A. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8641-

Gearing, D. P., Comeau, M. R., Friend, D. J., Gimpel, S. D., Thut, C. J., 8645

McGourty, J., Brasher, K. K., King, J. A., Gillis, S., Mosley, B., Ziegler, S. F., and Cosman, D. (1992) Science 255,1434-1437

Gearing, D. P. (1993) Adu. Immunol. 53,31-58 Zarling, J. M., Shoyab, M., Marquardt, H., Hanson, M. B., Lioubin, M. N., and

Brown, T. J., Rowe, J. M., Liu, J., and Shoyab, M. (1991) J. Immunol. 147, Todaro, G. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9739-9743

Richards, C. D., Brown, J. T., Shoyab, M., Baumann, H., and Gauldie, J. (1992) 2175-2180

Miles, S. A,, Martinez-Maza, O., Rezai, A., Magpantay, L., Kishimoto, T., Na- J. Immunol. 148, 1731-1736

Gearing, D. P., and Bruce, G. A. (1992) New Biol. 4 , 6 1 4 5 kamura, S., Radka, S. F., and Linsley, P. S. (1992) Science 255, 1432-1434

Liu, J., Modrell, B., Aruffo, A,, Marken, J. S., Taga, T., Yasukawa, K., Mu- rakami, M., Kishimoto, T., and Shoyab, M. (1992) J. Biol. Chem. 267,

Hibi, M., Murakami, M., Saito, M., Hirano, T., Taga, T., and Kishimoto, T. 16763-16766

(1990) Cell 63, 1149-1157 Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgen-

besser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopou- los, N. (1991) Cell 65, 663475

Ahn, N. G., Weiel, J. E., Chang, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494

Bird, T. A., Sleath, P. R., deRoos, P. C., Dower, S. K., and Virca, G. D. (1991 1 J . Biol. Chem. 266,22661-22670

Welham, M. J., Duronio, V., Sanghera, J. S., Pelech, S. L., and Schrader, J. W. (1992) J. Immunol. 149, 1683-1693

Van Lint, J., Agostinis, P., Vandervoorde, V, Haegeman, G., Fiers, W., Mer- levede, W., and Vandenheede, J. R. (1992) J. Bid. Chem. 266,2591G25921

Thomas, G. (1992) Cell 68, 3 4 Gearing, D. P., Thut, C. J., VandenBos, T., Gimpel, S. D., Delaney, P. D., King,

J., Price, V., Cosman, D., and Beckman, M. P. (1991) EMBO J. 10, 2839- 2848

Moreau, J.-F., Donaldson, D. D., Bennett, F., Witeck-Giannotti, J., Clark, S. C., and Wong, G. G. (1988) Nature 336,690-692

Kronheim, S. R., Cantrell, M. A,, Deeley, M. C., March, C. J., Glackin, P. J., Anderson, D. M., Hemenway, T., Merriam, J . E., Cosman, D., and Hopp, T.

Bird, T. A., Schule, H. D., Delaney, P. B., Sims, J. E., Thoma, B., and Dower, S. P. (1986) BioTechnology 4, 107%1082

Park, L. S., Friend, D., Grabstein, K., and Urdal, D. L. ( 1987) Proc. Natl. Acad. K. ( 1992) Cytokine 4, 429440

Benjamin, D., and Dower, S. K. (1990) Blood 75, 2017-2023 Sei. U. S. A. 84, 1669-1673

Thoma, B., Grell, M., Plizenmaier. K., and Scheurich. P. (1990) J. EXD. Med. 172, 1019-1023

Hannum. C. H.. and Thomoson. R. C. (1990) Nature 343.342-345 Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T.,

. I ~ ~~~

Payne, D. M . , Rossomando, A. J., Martino, P., Erickson, A. K., Her, J. H., Shabanowitz, J., Hunt, D. F., Weber, M. J., andSturgill, T. W. (1991)EMBO

Slack, J., McMahan, C. J., Waugh, S., Schooley, K., Spriggs, M. K., Sims, J. E., J. 10, 885-892

and Dower, S. K. (1993) J. Bid. Chem. 268, 2513-2524 Heldin, C.-H. (1992) EMBO J. 11, 42514259 Sakamaki, K., Miyajima, I., Kitamura, T., and Miyajima, A. (1992) EMBO J.

fakeshita, T., Asao, H., Obtani, K., Ishii, N., Kumaki, S., Tanaka, N., Mu- l l , 35413549

Davis, S., Aldrich, T. H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N. Y., nakata, H., Nakamura, M., and Sugamura, K. (1992) Science 257,379-382

and Yancopoulos, G. D. (1993) Science 260, 1805-1808

I ~~~ ~~-

6222 Differential Signal Dansduction by OSM Receptors 31. Wang, X., Moller, C., Norstedt, G., and Carter-Su, C. (1993) J. Eiol. Chem. 269,

35733579 32. Banner, D. W., DArcy, A,, Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C.,

Luetscher, H., and Lesslauer, W. (1993) Cell 73,431445 33. Murakami, M., Hibi, M., Nakawaga, N., Nakawaga, T., Yasukawa, K, Yamani-

shi, K., Taga, T., and Kishimoto, T. (1993) Science UU), 180%1810 34. Hilton, D. J., Nicola, N. A,, and Metcalf, D. (1991) J. Cell Physwl. 146,207-215 35. Linsley, P. S. , Bolton-Hanson, M., Horn, D., Malik, N., Kallestad, J. E., Ochs,

36. Sims, J. E., Gayle, M. A,, Slack, J. L., Alderson, M. R., Bird, T. A., Gin, J. G., V., Zarling, J. M., and Shoyab, M. (1989) J. Biol. Chem. 264,4282-4289

Colotta, E , Re, E , Mantovani, A., Shanebeck, K, Grabstein, K. H., and Dower, S . K. (1993) Proc. Natl. h a d . Sci. U. S. A. 90,6155-6159

37. Layton, M. J., Cross, B. A., Metcalf, D., Ward, D. L., Simpson, R. J., and Nicola, N. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,86164620

38. Baumann, H., Ziegler, S . F., Mosley, B., Morella, K. K., Pajovic, S. , and Gear- ing, D. P. (1993) J. Eiol. Chem. 268,8414-8417

39. Grell, M., Scheurich, P., Meager, A,, and Mzenmaier, K (1993) Lymphokine

40. Tartaglia, L. A., Weber, R. F., Figari, I. S., Reynolds, C., Palladino, M. A,, Jr., Cytokine Res. 12, 143-148

41. Eriksson, A., Siegbahn, A., Westermark, B., Heldin, C.-H., and Claesson- and Goeddel, D. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 88,9292-9296

42. L'allemain, G., Sturgill, T. W., and Weber, M. J. (1991) Mol. Cell. Eiol. 11, Welsh, L. (1992) EMEO J. 11, 543-550

43. Wood, K W., Sarnecki, C., Roberts, T. M., and Blenis, J. (1992) Cell 68, 1041- 1002-1008

44. Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and 1050

45. Kyriakis, J. M., App, H., Zhang. X.-f., Banejee, P., Brautigan, D. L., Rapp, U. Johnson, G. L. (1993) Science 260,315319

46. Schieven, G. L., Kallestad, J. C., Brown, T. J., Ledbetter, J. A., and Linsley, P. R., and Avruch, J. (1992) Nature 358, 417421

47. Baumann, H., Marinkovic-Pajovic, S., Won, K A., Jones, V. E., Campos, S. P., S. (1992) J. Immunol. 149, 167G1682

Jahreis, G. P., and Morella, K. K (1992) CIEA Found. Symp. 167,10&124

oncostatin m and leukemia inhibitory factor trigger overlapping

Documents