The regulation of gp130, leukemia inhibitory factor receptor and

Oncostatin M regulates synthesis and turnover of gp130,

leukemia inhibitory factor receptor α and oncostatin M

receptor β by distinct mechanisms*.

Frédéric Blanchard‡§║, Yanping Wang‡, Erin Kinzie‡, Laurence Duplomb¶, Anne

Godard¶, and Heinz Baumann‡§.

‡Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo,

New York 14263, and ¶INSERM U463, Institut de Biologie, 9 Quai Moncousu, 44035

Nantes Cedex 01, France.

Running title: Turnover of IL-6 cytokine receptors.

Key words: cytokine receptor, signal transduction, gene regulation, degradation.

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Footnotes:

*This work was supported by NIH grants CA 85580 and DK 38866 to HB, and Roswell

Park Cancer Support Grant CA16056. FB is a recipient of a fellowship from the

Association pour la Recherche contre le Cancer (ARC).

§To whom correspondence should be addressed: Frederic Blanchard or Heinz Baumann

at Roswell Park Cancer Institute, Department of Molecular and Cellular Biology, Buffalo

NY 14263. Phone: (716) 845-8175; Fax (716) 845-8389;

[email protected].

║Present address: INSERM U463, Institut de Biologie, 9 Quai Moncousu, 44035 Nantes

Cedex 01, France; E-mail: [email protected].

1The abbreviations used are: LIF, leukemia inhibitory factor; IL, interleukin; OSM,

oncostatin M; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-

regulated kinase; STAT, signal transducers and activators of transcription; PI3K,

phosphatidylinositol 3-kinase; G-CSF, granulocyte colony-stimulating factor; SHP1/2,

Src homology 2 domain-containing protein-tyrosine phosphatase; SOCS, suppressor of

cytokine signaling; JAK, Janus kinase; RIPA, radioimmune precipitation; GFP, green

fluorescent protein; APP, acute phase protein; RT-PCR, reverse transcription-polymerase

chain reaction.


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ABSTRACT

The cytokine receptor subunits gp130, leukemia inhibitory factor receptor α

(LIFRα) and oncostatin M receptor β (OSMRβ) transduce OSM signals that regulate

gene expression and cell proliferation. After ligand binding and activation of the

JAK/STAT and MAPK signal transduction pathways, negative feedback processes are

recruited. These processes attenuate receptor action by suppression of cytokine signaling

(SOCS) and by down-regulation of receptor protein expression. This study demonstrates

that in human fibroblasts or epithelial cells, OSM first decreases the level of gp130,

LIFRα and OSMRβ by ligand-induced receptor degradation, and then increases the level

of the receptors by enhanced synthesis. The transcriptional induction of gp130 gene by

OSM involves STAT3. Various cell lines expressing receptor subunits to the different IL-

6 class cytokines revealed that only LIFRα degradation is promoted by activated ERK,

and that degradation of gp130, OSMRβ and a fraction of LIFRα involves mechanisms

that are separate from signal transduction. These mechanisms include ligand-mediated

dimerization, internalization, and endosomal/lysosomal degradation. Proteosomal

degradation appears to involve a fraction of receptor subunit proteins that are

ubiquitinated independently of ligand binding.

INTRODUCTION

Interleukin-6 (IL-6)1, oncostatin M (OSM) and leukemia inhibitory factor (LIF)

are functionally and structurally related, and are part of the IL-6 family of cytokines (1-

5). Each IL-6 cytokine is recognized by a specific ligand-binding receptor subunit. In


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humans, OSM is exceptional in that it interacts with gp130 and with either LIFRα, or

OSMRβ to form the high affinity, signaling-competent OSM receptor complex I or II (3,

4). Ligand-induced oligomerization of receptor subunits activates Janus protein tyrosine

kinases (JAKs), which in turn phosphorylate tyrosine residues in the receptor cytoplasmic

domain. These phosphorylated tyrosines create docking sites for STAT transcription

factors (STAT1, 3 and 5), protein tyrosine phosphatase SHP-2, and linker proteins such

as Gab-1, Grb2 or SHC, which propagate the signal to other pathways (ERK 1/2, JNK,

PI3K; refs. 1-8). Receptor signaling is manifested by the activation of genes such as acute

phase proteins (APPs; ref. 2), or the cyclin-dependent kinase inhibitor p21WAF1 that is

primarily activated through STATs (9) and immediate early response genes such as c-fos,

c-jun and egr-1 primarily through ERK 1/2 (6).

Signaling by IL-6 cytokine receptors is transient, often restricted temporally and

in magnitude by the action of negative regulators. The SH2 domain-containing protein-

tyrosine phosphatases SHP1 and 2, through their catalytic function, attenuate the activity

of receptor-associated JAKs and consequently lower the induction of STAT-dependent

genes (4, 6). The suppressor of cytokine signaling SOCS1 and 3 are rapidly induced by

IL-6 cytokines and, through their SH2 domain, interact and deactivate JAKs or gp130 (4,

10). The protein inhibitors of activated STATs or PIAS associate with activated STATs

leading to a loss of STAT-DNA binding activity (11). Containment of IL-6 cytokine

signaling appears to be directed by two distinct mechanisms: (a) the induction or

mobilization of factors that attenuate functions of the cytoplasmic domains of the

receptor proteins and (b) the enhanced degradation of receptor proteins. Recently, we

have also demonstrated that receptor signals acting in trans determine the level of the


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receptor subunit LIFRα, and thus the cellular responsiveness to LIF (12). ERK 1/2,

activated by IL-6 cytokines or growth factors, phosphorylate serine 1044 (or serine 185

of the cytoplasmic domain) of LIFRα, leading to its lysosomal degradation independent

of LIF binding (12, 13). An additional ERK-independent degradation pathway for LIFRα

has also been observed in NIH-3T3 cells but this degradation occurred only following

LIF treatment (12). The other receptor subunits, gp130 and OSMRβ, do not possess a

phosphorylation site for ERK 1/2, and thus do not appear to be appreciably influenced by

activated ERK. However, serine 782 of gp130 located in the cytoplasmic domain has

been described as being phosphorylated and directing the cell surface expression of the

receptor subunit (14). The kinase for this modification is still unknown. Serine 782 is

located immediately N-terminal to the di-leucine motif of gp130 which was reported to

trigger the constitutive, ligand-independent endocytosis of gp130 (15). The adaptor

protein AP2 was noted to interact with the di-leucine motif, enabling the transfer of

receptors to clathrin-coated pits, endocytosis, and intracellular targeting to lysosomal

degradation (15, 16). No corresponding information regarding turnover of the other

receptor subunit OSMRβ is available.

In this study we asked whether ligand-dependent degradation of gp130 is indeed

determined by specific elements in its cytoplasmic domain, and whether turnover of

receptor complexes that include LIFRα and OSMRβ follow processes that apply to

gp130.

MATERIALS AND METHODS


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Tissue Culture Cells - NIH-3T3 fibroblasts, MCF7 breast carcinoma cells, and clonal

lines of H-35 cells stably expressing LIFRα (12), OSMRβ (8), the chimeric and

carboxyterminally FLAG epitope-tagged G-CSFR-gp130 construct with full length 277-

residues cytoplasmic domain of gp130 (6), or truncated G-CSFR-gp130(133)wt

(containing the 133-residue membrane-proximal cytoplasmic domain of gp130) or the

tyrosine-to-phenylalanine mutants of this chimeric construct, Y2F (Tyr759, or tyrosine 117

of the cytoplasmic domain), Y3F (Tyr767, or tyrosine 125 of the cytoplasmic domain), or

Y2,3F (tyrosines 117 and 125) (6,17) were maintained in DMEM containing 10% FCS

and antibiotics. Primary cultures of human pulmonary fibroblasts and alveolar epithelial

cells were prepared from residual lung tissues derived from surgical pneumectomy

specimens and provided by the Tissue Procurement Service at Roswell Park Cancer

Institute. The proliferating epithelial cells were maintained in serum-free keratinocyte

medium supplemented with cholera toxin and epidermal growth factor (GIBCO Life

Sciences). The homogeneity of the primary cell cultures was confirmed by

immunochemical staining for cell type-specific cytokeratins and integrins. To analyze

signaling or receptor down-regulation, cells were incubated for 5-18 h in serum-free

medium and then treated with 100 ng/ml recombinant IL-6 and LIF (Genetic Institute,

Cambridge, MA), human OSM (Immunex Corp., Seattle, WA), or mouse OSM (prepared

in COS-1 cells as described in ref. 8). MEK-1 activity was inhibited by 25 µM U0126

(Promega, Madison, WI), protein synthesis by 10 µM cycloheximide (Sigma), lysosome

activity by 100 µM chloroquine (Sigma), and proteasome activity by 1 µM MG132

(Calbiochem, La Jolla, CA).


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Plasmid Constructs - The following expression vectors have been described previously:

wild type and S185A mutant of human LIFRα (12, 13), human OSMRβ (8), human gp130

and chimeric human G-CSFR-gp130 (6) in the vector pDC302, and rat STAT3∆55C

(lacking 55 C-terminal residues; ref. 18) in pSV-Sport 1. Ubiquitin-HA in the expression

vector PMT123 was provided by Nicholas Heintz (University of Vermont; ref. 19). The

chimeric construct G-CSFR-gp130 with deleted cytoplasmic domain, but retaining the

transmembrane domain of gp130 (residues 599-645 of gp130) and with the Flag epitope

following the remaining 4 cytoplasmic residues (G-CSFR-gp130(∆cyto)Flag), was

generated by polymerase chain reaction (PCR) using the G-CSFR-gp130 in pDC302 as a

template. The chimeric receptor construct was transferred into the retroviral vector MINV

(6, 8). The retroviruses produced in the packaging PA317 cells were used to transduce H-

35 cells. Stable integrants were selected in medium containing 2 mg/ml G418 (6, 8).

Clonal lines expressing G-CSFR-gp130∆cyto-Flag were identified by immunoblotting for

the Flag epitope.

Transient Transfection - NIH-3T3 and MCF7 cells were transfected with FuGene6

(Roche Molecular Biochemicals) according to the manufacturer’s recommendation using

a ratio of 6 µl of FuGene6 to 4 µg of DNA. In all transfections, 0.25 µg of pEGFP(N1)

(Upstate Biotechnology Inc.) was included as marker of transfection efficiency (8). To

enrich for NIH-3T3 cells transfected with expression vectors for STAT3∆55C, GFP- and

GFP+ cells were selected by sterile fluorescence-activated cell sorting as described (8).

Immunoprecipitation and Western Blotting - Cell monolayers were lysed in RIPA buffer

(50 µl per cm2 monolayers). Lysates were incubated with antibodies and protein-G-

conjugated Sepharose (Amersham Pharmacia Biotechnology). The immunoprecipitates or


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aliquots of whole cell lysates were separated on 6% to 12% SDS-polyacrylamide gels and

proteins were transferred to protean membranes (Schleicher & Schuell). The membranes

were reacted with antibodies to the extracellular domain of human OSMRβ (Immunex

Corp.), to the carboxyterminal peptide of the cytoplasmic domain of LIFRα or gp130

(Santa Cruz Biotechnology), to STAT3, ERK 1/2, SOCS3, SHP-2, Flag, HA (Santa Cruz

Biotechnology), PY-STAT3, PY-STAT5, P-p38, P-ERK (New England Biolabs, Inc.),

JAK1, Myc (Upstate Biotechnology, Inc.) and followed with secondary antibodies (ICN

Biomedicals, Inc., Aurora, OH) in PBS containing 0.1% TWEEN, 5% milk or 3% BSA.

Immunoreactions were visualized by enhanced chemiluminescence reaction (ECL)

according to the manufacturer (Amersham Pharmacia Biotech). From each blot, several

x-ray films were prepared by exposing for different length of times. The bands on these

films were scanned by densitometry and quantified by using the ImageQuant program

(Molecular Dynamics).

RT-PCR and Northern Blot analysis - Cellular RNA was extracted by the Trizol method

(Life Technology, Grand Island, NY). For RT-PCR analysis, aliquots of 8 ng to 5 µg of

RNA were subjected to cDNA synthesis with 400 U of M-MLV reverse transcriptase

(GIBCO/BRL) and 0.5 µg oligo(dT) 15-mer. The cDNA present in 5 µL of reaction

mixture was amplified with 0.625 U of Taq polymerase (Promega) and 10 pmol each of

sense and antisense primers (20, 21). The thermal cycle profile was as follows:

Denaturation for 1 min at 94°C, annealing for 1 min at 59°C and extension for 1 min at

72°C, for 30 to 35 cycles. The ethidium bromide-stained patterns of the

electrophoretically separated PCR products were digitally photographed. The staining

intensity of the bands was determined by integration of the pixel values using the


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ImageQuant program. For northern Blot analysis, aliquots of 20 µg of RNA were

separated on 1.5% formaldehyde-agarose gel, transferred to nylon membrane (Schleicher

& Schuell), and reacted with 32P-labeled cDNA probes for OSMRβ or gp130. The

radioactive patterns were visualized by autoradiography and by phosphorimaging

(Molecular Dynamics). The ethidium bromide staining pattern of separated ribosomal

RNAs was used as a marker for sample loading.

RESULTS

Modification of receptor subunits level during OSM treatment.

Recently, we showed that rat H-35 hepatoma cells respond to 6 h treatment with

LIF or OSM by down-regulation of LIFRα (12). In contrast, the level of gp130 remains

essentially constant with only a slight decrease during the first hour and return to basal

value by 2 h (12). To identify whether the mode of receptor subunit turnover as

determined in hepatoma cells is also established in other, non-hepatic and non-

transformed cell types, we analyzed normal human lung fibroblasts and epithelial cells.

Based on the activation profile of STAT3, STAT5, and MAP kinases p38 and ERK 1/2,

lung fibroblasts responded strongly to OSM and to a lesser degree to LIF or IL-6 (Fig.

1A). Similar results have been obtained with bronchial and alveolar epithelial cells (data

not shown; refs. 22, 23). Within 2 hours of OSM treatment, the level of activated STAT3

was reduced by 90 % in both fibroblasts and epithelial cells, but by 4 to 6 hours of

treatment it increased again (Fig. 1B and D). During this treatment period, the level of the

fully processed form of OSMRβ (labeled ‘’Form 1’’ in Fig. 1B) was reduced to 40%,

whereas the level of the precursor form (labeled ‘’Form 2’’) was increased two-fold. In


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the same cells, the higher molecular forms of LIFRα and gp130 (“Form 1”) decreased to

50% by a 2 h OSM treatment, followed by recovery of the original level (Fig. 1B). The

levels of LIFRα and gp130 precursor forms (“Form 2”) were increased approximately

two-fold during the 6 h OSM treatment period (Fig. 1B and C). None of these changes in

receptor expression were evident in cells treated with LIF or IL-6, probably because of

the relative low-level activity of corresponding receptors in these cells (Fig. 1A, and data

not shown). Interestingly, in fibroblasts and epithelial cells, an induced expression of

SOCS3 could be detected by immunoblotting that peaked at 1 h of OSM treatment (Fig.

1B and D, lanes marked “60”). These results suggest that receptor subunits follow

specific turnover mechanisms that are characterized by an initial boost of ligand-induced

down-regulation of receptor protein, followed by a stimulated synthesis. Together with

induced signal-modifying factors, such as SOCS3, the regulated expression of receptor

proteins appear to determine the temporal profile of activated STAT3 in long-term OSM-

treated cells.

Receptors mRNA levels are induced by OSM treatment.

To address the mechanisms by which OSM induces receptors levels, we first

analyzed levels of receptors mRNA by Northern blotting for lung fibroblasts (Fig. 2A)

and epithelial cells (not shown). Due to technical limitations, only OSMRβ, but not

LIFRα or gp130, yielded a significant signal above background with this technique. The

results indicate an increase of OSMRβ mRNA by 6 h of OSM treatment, and elevated

levels maintained for at least 24 h. To assess the effect of OSM on mRNA of the other

receptor subunits, transcripts were analyzed by RT-PCR. RT-PCR with serial dilutions of


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the input total RNA suggested a higher abundance for gp130 mRNA than OSMRβ and

LIFRα mRNA (Fig. 2B). After 24 h OSM treatment, the signal for OSMRβ mRNA was

increased 7-fold, LIFRα mRNA was increased 5-fold, and gp130 mRNA was increased

3-fold (Fig. 2C and D). An immediate, but partly transient induction of CIS, SOCS1 and

SOCS3 mRNAs was detected that peaked at 1-2 h of treatment.

Induction of receptor subunits degradation and synthesis by OSM is a general

mechanism.

The screening of various established cell lines from hepatic, mesenchymal, and

epithelial origins confirmed the general features of receptor level modulation by OSM.

Among these cell lines, we identified NIH-3T3 fibroblasts as a prominent target for

induction of gp130 expression by OSM treatment, but as in primary fibroblasts, not by

LIF or IL-6 (Fig. 3A; data not shown for IL-6). Furthermore, like human fibroblasts, 3T3

cells displayed a similar kinetic of STAT3 activation by OSM. The reduction of activated

STAT3 temporally correlated with OSM-induced SOCS3 (Fig. 3A). However, after a 2 h

OSM treatment, the level of activated STAT3 increased again, concomitant with a rise in

gp130 mRNA (Fig. 3D) and protein (Fig. 3A and B). As noted previously (12), OSM

treatment produces a transient decrease of gp130 protein within the first 30 min of

treatment (Fig. 3B and C) whereas LIFRα was reduced to 10% level and stayed low

during the continued OSM treatment. Due to lack of suitable antibodies against mouse

OSMRβ, the level of OSMRβ in 3T3 cells could not be determined. The fact that OSM

treatment did elicit a ligand-dependent, continuous degradation of gp130 parallel to

enhanced synthesis is recognized by the accumulation of gp130 fragments, representing


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gp130 without extracellular domain that is detectable by the antibodies directed against

the C-terminal epitope of gp130 (Fig. 3B, lower panel). In order to assess the degradation

of receptor protein separate from biosynthesis, 3T3 cells were treated in presence of

cycloheximide (Fig. 3A). The half-life of LIFRα and gp130 was determined to be

between 90 to 120 min, and this was reduced to 60 min in presence of OSM (Fig. 3A and

C). As expected, the induction of SOCS3 protein expression was inhibited by

cycloheximide treatment (Fig. 3A). Furthermore, in the same cells, the reappearance of

activated STAT3 after 120 min of OSM treatment was prevented, suggesting that this

fraction of activated STAT3 in cells not treated with cycloheximide depends on newly

synthesized STAT3 and/or receptors. The data presented thus far (Figs. 1-3) suggest that

LIFRα, OSMRβ and gp130 are targeted for degradation after OSM treatment, and that an

effective compensatory synthesis of OSMRβ, gp130, and to a lesser extent LIFRα, occurs

that correlates with sustained STAT3 activation after OSM treatment but not after

treatment with LIF.

Induction of gp130 synthesis by OSM is mediated by STAT3.

The STAT3 and ERK 1/2 pathways are two of the major signaling pathways

activated by OSM. To determine their relative contribution to the induction of gp130

synthesis, NIH-3T3 cells were treated with the MEK-1 inhibitor, U0126, or transfected

with an expression vector encoding the dominant negative form of STAT3, STAT3∆55C

(18). As shown in Fig. 4, U0126 inhibited the activation of ERK 1/2 by OSM, but did not

modify the activation of STAT3. Induction of gp130 was maintained in U0126-treated

cells, but induction of SOCS3 was reduced by 50%. In contrast, in STAT3∆55C-


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overexpressing cells, gp130 was not increased after OSM treatment and SOCS3 showed

attenuated induction.

A functional, cis-acting binding site for activated STAT3 has been reported within

the gp130 promoter (24). Therefore, we cloned this gp130 promoter in the pCAT vector,

and analyzed the effects of OSM treatment on the activity of this promoter. We observed

that, depending on the cell line used, OSM induced 2- to 10-fold the activity of the gp130

promoter, and this induction was prevented by transfection with STAT3∆55C (data not

shown). Together these results strongly suggest that the increased gp130 mRNA and

protein in OSM-treated 3T3 cells (Fig. 3 and 4) result from the transcriptional activation

of gp130 gene by STAT3. SOCS3 gene induction appears to depend on STAT3 and ERK

pathways.

Down-regulation of gp130 after ligand binding does not depend on cytoplasmic

motif.

Previous studies have suggested that di-leucine motifs and serine phosphorylation

in the gp130 cytoplasmic domain direct degradation of gp130 and LIFRα (12-16). To

define the requirement of cytoplasmic domain elements for the ligand-induced

degradation of receptor subunit, we took advantage of H-35 cells with stable expression

of transduced epitope tagged LIFRα, OSMRβ, and chimeric G-CSFR-gp130 (6, 8, 12,

17). In those cell lines, the integrated viral vector is under the transcriptional control of

the viral LTR promoter, and its expression is not influenced by cytokine treatment. In

OSMRβ-transduced H-35 cells, the endogenous LIFRα was downregulated by LIF (Fig.

5Aa) and also by OSM treatment (Fig. 5Ab). Similarly in G-CSFR-gp130 expressing


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cells, G-CSF treatment through gp130 signaling was effective in reducing LIFRα (Fig.

5Ac). In both systems, this action in trans can be prevented by incubation with the MEK-

1 inhibitor U0126 (Fig. 5Aa-c). The conditions of U0126 treatment were such that

STAT3 activation by cytokines was maintained at normal levels, but ERK activation was

essentially absent (Fig. 5Ad). In contrast to LIFRα, OSMRβ or G-CSFR-gp130 down-

regulation was observed only after OSM or G-CSF treatment, respectively, and was not

affected by U0126 (Fig. 5Aa-c). Similarly, treatment with IL-6, insulin, or PMA induced

down-regulation of LIFRα in trans, but not of OSMRβ or G-CSFR-gp130 (Fig. 5B). As

described previously (12), LIFRα with mutation of the ERK substrate site, Ser185 to Ala,

was no longer subject to down-regulation by ERK action in trans. However, this mutant

LIFRα retained a LIF inducible decrease (Fig. 5B). These results suggest that LIFRα,

OSMRβ and gp130 are downregulated by ligand binding, and that only LIFRα is subject

to an additional, ERK-sensitive and Ser185-dependent degradation mechanism.

To characterize the mechanism underlying the prominent down-regulation of

gp130, we established H-35 cell lines expressing specific and C-terminally Flag epitope-

tagged G-CSFR-gp130 forms with truncated cytoplasmic gp130 domains (schematically

shown in Fig. 6). These chimeric receptors contain the 133-residue, membrane-proximal

domain of gp130 (6.17) and, hence, do not include any of the proposed elements

specifically directing endocytosis and degradation. Mutant forms of the chimeric receptor

were designed that recruited specifically JAKs, STAT3, and/or SHP2/ERK. The

engagement of the STAT3 pathway was eliminated by mutating the tyrosine residue

within the single remaining STAT3 binding element (Box3) at position 125 of the

cytoplamic domain (= Y3F; ref 17). The recruitment of the SHP2/ERK pathway was


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suppressed by mutating the SHP2 binding element at position 117 (=Y2F; ref 6).

Mutations at both sites (= Y2,3F) generated a receptor limited to the activation of JAKs..

All these chimeric receptors displayed an equal down-regulation after dimerization

directed by G-CSF treatment, regardless of the activation of signaling molecules. This

finding suggests: (a) the di-leucine motif or Ser782 implicated in internalization and cell

surface expression, respectively, are not required for the ligand-induced down-regulation

of receptor protein; (b) cells expressing the G-CSFR-gp130(Y2F) mutant are devoid of

SHP-2 recruitment and display a sustained activation of STAT3. This enhanced STAT

action is not a function of higher levels of receptors or JAK phosphorylation, but is likely

due to the loss of inhibitory activity of SHP-2 (6); (c) cells with G-CSFR-gp130(Y3F)

mutant show only a low level of activated STAT1 and 3 (see Fig. 6, EMSA, bottom

panel), but a magnified phosphorylation of SHP2 and ERK; and (d) the G-CSFR-gp130

construct with a deleted cytoplasmic domain, G-CSFR-gp130(∆cyto), is turned over with

the same kinetic as the G-CSFR-gp130(Y2,3F) that retained a strong JAK1 activation.

This suggests that JAK action is not critical for receptor degradation but that extracellular

and transmembrane domains are sufficient to direct the ligand-induced degradation.

Lysosomal and proteosomal degradation of receptor subunits.

We next determined the relative contribution of different degradation pathways to

receptors turnover as part of ligand-induced down-regulation. Treatment of primary

fibroblasts for 6 hours with the proteosome inhibitor MG132, or the lysosome inhibitor

chloroquine, alone or together with cycloheximide, indicated an increased half-life of

both LIFRα and gp130 (Fig. 7A). This implies that proteosomal and lysosomal pathways


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contribute to the normal turnover of the receptor subunits. Chloroquine treatment also

induced an accumulation of gp130 degradation products (Fig. 7A, “gp130 fragments”;

fragments derived from LIFRα are not shown; see ref. 12). MG132 and chloroquine have

a more important effect on LIFRα than gp130, as evident from the several-fold higher

level of receptor protein after 6 h of treatment with these drugs (Fig. 7A, lanes marked

with 0 min cycloheximide treatment). An equivalent involvement of the two degradation

pathways in G-CSF-induced G-CSFR-gp130 down-regulation is demonstrated in G-

CSFR-gp130 transduced cells treated with MG132 or chloroquine (Fig. 7B).

To better define the role of ubiquitination as part of the proteosomal degradation

of receptor subunits, we assessed the level of ubiquitination of gp130, OSMRβ and

LIFRα. Expression vector for these receptor subunits, together with that for HA-tagged

ubiquitin, were transfected in MCF7 cells (19). The receptor subunits were

immunoprecipitated and the presence of ubiquitin was identified by HA immunoblotting.

Based on the pattern of immunodetectable signals in Fig. 7Ca-c, immunoprecipitated

gp130, OSMRβ and LIFRα appeared to include poly-ubiquitinated species. Transfected

STAT3 served as a control for an over-expressed protein that was not affected by

ubiquitin (Fig. 7Cd). Treatment with corresponding ligands did not modify the intensity

of receptor ubiquitination, but treatment with MG132 caused an accumulation of

ubiquitinated receptor subunits. This suggests that a fraction of gp130, OSMRβ and

LIFRα in the cells is ubiquitinated independently of cytokine treatment and that this

fraction may be a target for proteosomal degradation.

DISCUSSION


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Numerous studies have addressed the mechanisms by which the signaling of

hematopoietic cytokines is restricted in time and magnitude. In the example of IL-6

cytokines, two basic processes have emerged. One process is the moderating action of

specific suppressors such as SHP1/2, SOCS1/3 and PIAS on signals activated by gp130,

LIFRα and OSMRβ (3, 4, 6, 10, 11). The other process is the adjustment of the

expression levels of receptor subunits and the regulation of down-stream signal

transduction pathways. Recent studies by us (12) and others (13, 14, 15) have identified

regulated degradation of receptor subunits as significant factors that determine cellular

responsiveness to IL-6 cytokines. We have demonstrated that differences in the control of

receptor turnover exist which affect individual receptor proteins. LIFRα is unique in that

phosphorylation by activated ERK 1/2 induces the lysososomal degradation of the protein

(12, 13). In the present study, we addressed the process by which the expression of the

more stable gp130 and OSMRβ is controlled. We determined that ligand binding

enhances degradation of these subunits, in part as noted for LIFRα, but that

compensatory mechanisms, through increased receptor synthesis, reestablish and

maintain levels of cytokine responsiveness close to pretreatment. We suggest that ligand-

induced synthesis is primarily mediated by a STAT3-dependent induction of the receptor

genes. The ligand-induced degradation of functional receptor proteins is not critically

dependent on JAKs, SHP2/ERK or STAT3 signaling, or any specific cytoplasmic domain

structure. We propose that ligand-induced dimerization of receptor subunits, through the

extracellular and transmembrane domains alone, initiates endocytosis, and that

degradation involves lysosomal as well as proteosomal, ubiquitin-mediated pathways.


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Regulated synthesis and degradation are effective mechanisms by which cells adjust their

IL-6 type cytokine responses.

Availability of cytokines and receptor expression represent two of the most

fundamental targets for determining cytokine responsiveness of cells. The next level of

importance includes control of cytokine receptor action as a function of cytokine

treatment. Two components for this control are identified here: (a) expression of receptor

subunits is inducible by the receptor signals; and (b) the ligand-recruited receptor

complex is tagged for degradation beyond the constitutive turnover process. The balance

of the two processes establishes the temporal profile of potential cytokine receptor action.

Previous analyses have indicated that IL-6 or OSM treatment increase gp130

promoter activity (24). Since a functional STAT-binding element is present and necessary

within the gp130 promoter to mediate this effect, it has been suggested that activation of

gp130 by its ligands may stimulate the production of new gp130 to replenish receptors

consumed upon ligand activation (24). Indeed, in normal lung fibroblasts and epithelial

cells as well as in various cell lines, we detected an increased production of gp130, but

also OSMRβ and, to a lower extent, LIFRα. Only OSM treatment appears to be able to

trigger these effects, correlating with the finding that OSM is a more effective inducer of

signaling than IL-6 or LIF in these cells (22, 23). Receptor protein levels increase

proportional to the mRNA for the gene products. Transfection of dominant negative

STAT3 into 3T3 cells confirmed the predicted transcriptional role of STAT3 for

regulation of the gp130 gene. Whether a similar STAT3-dependent mechanism is

responsible for ligand-induced synthesis of OSMRβ and LIFRα in normal fibroblasts

(Fig. 1-2) remained to be determined.


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LIFRα, gp130, and JAKs have half-lives of 2-4 hours (12, 25). In contrast,

STATs and SHP2 have a slower turnover rate and SOCS1/3 are very short-lived (12, 25).

In fact, the immunodetectable level of these proteins is not always a direct reflection of

their functional contribution to the cytokine effects. Although the signal-mediating

molecules STATs and SHP2 are usually expressed at relatively high levels, only that

fraction of protein physically recruited by receptors is functionally relevant, and the

activity of these proteins is strictly regulated by post-translational modification (i.e.

phosphorylation). SOCS proteins, the functions of which are not strictly directed by post-

translational modifications, are largely regulated at the transcriptional level by, among

other factors, activated STATs (Fig. 4; ref. 3, 10, 25). Receptor subunits appear to be

regulated by two processes: (a) immediately after initial ligand binding, the activity of

most, if not all cell-surface exposed receptors is induced by phosphorylation, unless there

is a limited amount of JAKs to react with all available receptor proteins; and (b)

subsequently, continued function of receptors is dependent on the rate of synthesis, which

in the case of gp130 includes a STAT specific stimulation of transcription, and on the rate

of degradation. We have previously demonstrated that LIFRα degradation is enhanced by

the ERK-dependent phosphorylation of LIFRα cytoplasmic domain at Ser185 (12). Since

phosphorylation of gp130 at Ser782 also regulates cell surface expression of gp130 (14), it

was conceivable that ligand-induced activation of cytoplasmic protein kinases mediates

Ser-phosphorylation of receptor subunits to regulate their turnover. However, we

observed that truncated gp130 with 133 residues of the cytoplasmic domain, hence

devoid of the Ser782-phosphorylation site, is still downregulated. This down-regulation is

compatible with that observed for LIFRα with the Ser185 to Ala mutation that is no longer


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the target of ERK-directed down-regulation (Fig. 5B). The study of truncated receptor

subunits also indicated that the di-leucine motifs (Leu786Leu787) implicated in receptor

internalization (15, 16, 29), and the sites for STAT, SHP2, and even for JAKs activation,

are dispensable for ligand-induced down-regulation of G-CSFR-gp130 (Fig. 6).

Therefore, we conclude that dimerization of receptor subunits without intracellular signal

transduction is sufficient to trigger the process of receptor degradation. Similar

conclusions were reached in other receptor systems: (a) monoclonal antibodies against

the EGF receptor act as inhibitors and induce receptor down-regulation only in their

dimerizing forms (26); and (b) a point mutation within the FGF receptor 3

transmembrane domain leads to a selective delay in the down-regulation and ligand-

induced internalization of the receptor (27).

Kinetic studies have revealed that IL-6 treatment does not appreciably modify the

rate of gp130 internalization, which is largely constitutive (3, 12, 16, 28). Since truncated

gp130 without the di-leucine motif and even without the cytoplasmic domain, are still

down-regulated after ligand binding, internalization mechanisms other than those

proposed to engage the di-leucine motif and AP-2 dependent processes remain effective.

Based on chloroquine experiments (Fig. 7, ref. 12), we have demonstrated that following

internalization, gp130 and LIFRα are targeted for lysosomal degradation, where intact

receptors as well as cytoplasmic receptor fragments, products of endoproteolytic release

of the extracellular domain, accumulate. Moreover, we have also observed an

accumulation of receptor subunits, with intact cytoplasmic domains, after inhibition of

proteosome activity. Therefore, it is conceivable that gp130, LIFRα and OSMRβ are

targets for proteosomal degradation by their direct ubiquitination, or by association with


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other ubiquitinated proteins, such as SOCS/CIS proteins (30, 31). The former hypothesis

is supported by the finding that gp130, LIFRα and OSMRβ are directly ubiquitinated and

ubiquitinated receptor proteins accumulate after proteosome inhibition (Fig. 7C). Similar

observations have been made previously with the growth hormone receptor (32, 33).

However, truncated gp130, with no cytoplasmic domain and no identifiable direct

ubiquitination, is still effectively downregulated after ligand binding, indicating that

gp130 down-regulation does not strictly depend on ubiquitin or proteosomal degradation.

Together, our results suggest that, in addition to other mechanisms reported to

determine the constitutive receptor degradation, ligand-induced dimerization enhances

gp130, LIFRα and OSMRβ turnover by a process that depends on di-leucine independent

internalization, endosomes to lysosomes trafficking, and/or lysosomal degradation.

Additionally, our data indicate that ERK-specific down-regulation of receptor protein is

limited to LIFRα. A compensatory mechanism retaining specific cytokine

responsiveness, is the enhanced synthesis of receptor subunits, especially gp130 and

OSMRβ.

Acknowledgments: We are grateful to Immunex Corporation for providing cloned

human cytokine receptors and anti sera against human OSMRβ, Immunex Corporation

and Genetic Institute for cytokines, Dr. A. Miyajima for the mouse OSM expression

vector, Dr G. Fey and Dr. J. Ripperger for the STAT3 expression vector, and to Dr. G.

Loewen for bronchial brushings to prepare epithelial cells.


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(1998) Biochemical J. 334, 297-314

4. Auernhammer, C.J., and Melmed, S. Leukemia-Inhibitory Factor. (2000) Endocrine

Reviews 21, 313-345

5. Elson, G.C., Lelievre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J.,

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Gascan, H. (2000) Nat. Neurosci. 3, 867-872

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7. Hermanns, H.M., Radtke, S., Schaper, F., Heinrich, P.C., and Behrmann, I. (2000)

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Silva, A., Asimakis, M., Farley, A., Nash, A.D., Metcalf, D., Hilton, D.J., Nicola,

N.A., and Baca, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6493-6498

11. Chung, C.D., Liao, J., Liu, B., Rao, X., Jay, P., Berta, P., and Shuai, K. (1997)

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12. Blanchard, F., Duplomb, L., Wang, Y., Robledo, O., Kinzie, E., Pitard, V., Godard,

A., Jacques, Y., and Baumann, H. (2000) J. Biol. Chem. 275, 28793-28801

13. Schiemann, W.P., Graves, L.M., Baumann, H., Morella, K.K., Gearing, D.P.,

Nielsen, M.D., Krebs, E.G., and Nathanson, N.M. (1995) Proc. Natl. Acad. Sci. U.

S. A. 92, 5361-5365

14. Gibson, R.M., Schiemann, W.P., Prichard, L.B., Reno, J.M., Ericsson, L.H., and

Nathanson, N.M. (2000) J. Biol. Chem. 275, 22574-22582

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and Graeve, L. (1998) FEBS Lett. 441, 231-234

17. Lai, C.F., Ripperger, J., Wang, Y., Kim, H., Hawley, R.B., and Baumann. H. (1999)

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20. Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J.L., Moreau,

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273, 20886-20893

21. Magrangeas, F., Boisteau, O., Denis, S., Jacques, Y., and Minvielle, S. (2001) Eur.

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23. Richards, C.D., Kerr, C., Tanaka, M., Hara, T., Miyajima, A., Pennica, D., Botelho,

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26. Fan, Z., Lu, Y., Wu, X., and Mendelsohn, J. (1994) J. Biol. Chem. 269, 27595-

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28. Thiel, S., Behrmann, I., Dittrich, E., Muys, L., Tavernier, J., Wijdenes, J., Heinrich,

P.C., and Graeve, L. (1998) Biochem. J. 330, 47-54

29. Thiel, S., Behrmann, I., Timmermann, A., Dahmen, H., Muller-Newen, G.,

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Biochem. J. 339, 15-19

30. Verdier, F., Chretien, S., Muller, O., Varlet, P., Yoshimura, A., Gisselbrecht, S.,

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31. Zhang, J.G., Farley, A., Nicholson, S.E., Willson, T.A., Zugaro, L.M., Simpson,

R.J., Moritz, R.L., Cary, D., Richardson, R., Hausmann, G., Kile, B.J., Kent, S.B.,

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Natl. Acad. Sci. U. S. A. 96, 2071-2076

32. Govers, R., ten Broeke, T., van Kerkhof, P., Schwartz, A.L., and Strous, G.J. (1999)

EMBO J. 18, 28-36


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33. Alves Dos Santos, C.M., ten Broeke, T., and Strous, G.J. (2001) J. Biol. Chem. Jun

19 [epub ahead of print]

FIGURE LEGEND

Fig. 1: OSM modulates the level of receptor subunits in normal fibroblasts and

epithelial cells. A, to establish the cytokine response profile of normal human pulmonary

fibroblasts, confluent cultures (passage 2) were treated for 15 min with 100 ng/ml LIF,

OSM or IL-6. Aliquots of whole cell lysates were then analyzed by immunoblotting for

phospho-STAT3 (Tyr705)(PY-STAT3), phospho-STAT5 (Tyr694/Tyr699)(PY-STAT5),

phospho-p38 MAPK (Thr180/Tyr182)(P-p38), and phospho-ERK (Thr202/Tyr204)(P-ERK).

B to D, pulmonary fibroblasts or epithelial cells were treated for 0 to 360 min with OSM,

and analyzed for the indicated receptor subunits, activated STAT3 (PY-STAT3), total

STAT3 and SOCS3 by immunoblotting. Signals corresponding to form 1 (fully

processed) and 2 (precursor form) of the receptor subunits were quantified by

densitometry and values in C were expressed relative to untreated control cells (=100%).

Fig. 2: OSM increases receptor mRNA levels. Human pulmonary fibroblasts were

treated for 0 to 24 h with OSM and cellular RNA was extracted. A, 20 µg-aliquots of

RNA were subjected to northern blot hybridization for mRNA encoding OSMRβ.

Ethidium bromide staining of rRNAs served as a measure for RNA loading. B, serial

dilutions of RNA from control cells were analyzed by RT-PCR with primers specific for

the indicated cDNA. C, aliquots of 0.2 µg RNA were used for RT-PCR of OSMRβ and


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LIFRα sequences, and 0.04 µg RNA for gp130. D, the fluorescence signals

corresponding to the RT-PCR products for the receptor mRNAs were quantified and

expressed relative to the value of the control cells.

Fig. 3: OSM modulates expression of gp130 and LIFRα in NIH-3T3 cells. A to C,

NIH-3T3 fibroblasts were treated with LIF, murine OSM, 10 µM cycloheximide (CHX)

or combinations for 0 to 360 min. Whole cell lysates were analyzed for the indicated

proteins by immunoblotting. For gp130 analysis, ECL reaction was exposed for 5 min in

A, and the same membrane containing the OSM-treated cell extracts was also exposed for

10 sec in B (upper section). Signals corresponding to gp130 were quantified by

densitometry and expressed relative to controls (=100%) in C. D, total cell RNAs were

extracted from NIH-3T3 cells treated with OSM for the indicated lengths of time and

analyzed by Northern blotting for gp130 mRNA.

Fig. 4: STAT3 mediates induction of gp130 by OSM. NIH-3T3 fibroblasts were

transfected with expression vector for GFP and C-terminal truncated STAT3 (STAT3∆).

From these cultures, populations of GFP- (control) and GFP+ (STAT3∆ transfected) cells

were enriched by fluorescence-activated cell sorting. Subcultures of GFP- and GFP+ cells

were treated with OSM in the presence or absence of 25 µM MEK-1 inhibitor (UO126).

Whole cell lysates were then analyzed for indicated proteins by immunoblotting.

Fig. 5: Down-regulation of receptor subunits in transduced H-35 cells. A, subclonal

lines of H-35 cells expressing murine OSMRβ (Aa and Ab) or G-CSFR-gp130(277) wild


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type (G-gp130; Ac) were treated with LIF, murine OSM or G-CSF for 0 to 120 min in the

presence or absence of 25 µM U0126. Whole cell lysates were then analyzed for

indicated receptor subunits by immunoblotting. The inhibitory effect of U0126 on ERK

activation but not STAT3 activation by 15 min LIF treatment of H-35 cells is presented

in Ad. B, H-35 cells expressing wild type LIFRα, mutant LIFRα(S/A), murine OSMRβ

or G-CSFR-gp130 were treated with indicated factors and analyzed by immunoblotting

for levels of the receptor proteins. Signals corresponding to higher molecular size forms

(Form 1) were quantified by densitometry and expressed relative to untreated cultures

(=100%).

Fig. 6: Ligand-induced down-regulation of G-CSF-gp130 does not depend on

specific cytoplasmic motif. Clonal lines of H-35 cells were used which express G-

CSFR-gp130, containing either 133 residues of the cytoplasmic domain of wild type

gp130, the indicated Y/F mutations of this construct, or deleted cytoplasmic domain (see

schematic representations). Cells were treated for 0 to 120 min with G-CSF and analyzed

by western blotting for the Flag tagged receptor proteins (top panels) or the signaling

proteins (middle panels). For the DNA mobility shift assay (EMSA, bottom panels), cell

extracts were incubated with a labeled probe corresponding to high affinity SIS-inducible

element (SIE). (ND, not determined).

Fig. 7: Down-regulation of receptor subunits depends on lysosomal and proteosomal

degradation. Aa, human pulmonary fibroblasts were treated for 360 min with medium

alone (Control) or with medium containing 1 µM proteasome inhibitor MG132 (MG) or


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100 µM lysosome inhibitor chloroquine ( “0 min” lane in each panel). Parallel cultures

were treated with these reagents in combinations with 10 µM cycloheximide for 120 to

360 min (CHX). Whole cell lysates were analyzed for full length LIFRα or full length

and proteolytic fragments of gp130 as indicated (NS, nonspecific). Ab, signals

corresponding to the higher molecular size forms of the full length receptor proteins

detected in the gel patterns of Aa were quantified by densitometry and expressed relative

to the values of the untreated control cultures. B, H-35 cells expressing G-CSFR-

gp130(277) wild type were treated with medium alone (Control), or with medium

containing MG132 (MG), chloroquine (Chl.) and G-CSF for the times indicated. Cell

lysates were analyzed by immunoblotting for the indicated proteins. C, MCF7 cells were

transfected with expression vector for HA-tagged ubiquitin (Ub-HA) together with

expression vectors for OSMRβ (Ca), LIFRα (Cb), gp130 (Cc), or STAT3 (Cd), or empty

vector (Control) as indicated at the top of each panel set. Cells were then treated with

OSM or LIF, and 1 µM MG132 for 6 h. Receptor subunits or STAT3 were

immunoprecipitated and analyzed by immunoblotting for ubiquitin-HA (upper panels)

and receptor subunit or STAT3 protein (lower panels).


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Fig. 1

0 15 60 120 240 360

LIFRα

gp130

PY-STAT3STAT3

BOSM:

OSMRβ

Lung Fibroblasts

Form 1Form 2

Form 1Form 2

Form 1Form 2

SOCS3

Form 1Form 2

0

50

100

150

200

050

100150200250

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100

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0 15 60 120 240 360OSM (min)

Rec

epto

r Lev

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of C

ontro

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COSMRβ

LIFRα

gp130

0 15 60 120 240 360Alveolar Epithelial Cells

PY-STAT3STAT3

OSMRβ

DOSM:

SOCS3

LIF OSMIL-

6

PY-STAT3

P-ERK

PY-STAT5

P-p38

Lung Fibroblasts

Contro

l

A

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0.00

8

0.04

0.2

OSMRβ

Total RNA (µg):B

A 0 1 2 4 6 24OSM (hours)

OSMRβ

EtBrStain

Fig. 2

0 1 2 4 6 24

01234567

012345

0

1

2

3

mR

NA

Sign

al (R

elat

ive

to C

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OSMRβ

LIFRα

gp130

OSM (hours)

0 1 2 4 6 24OSM (hours)

OSMRβ

LIFRα

gp130

SOCS1

SOCS3

CIS

Actin

C D

LIFRα

gp130

Actin


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Fig. 3

gp130

gp130 Fragments

ECL 10 sec

ECL 5 min

210

122

80

52

36

0 15 60 120 240 360OSMB

0

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200

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OSMCHXOSM+CHX

gp13

0 Le

vel (

% o

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Time (min)

C

0 15 60 360 0 15 60 120 240 360 0 15 60 120 240 360 0 15 60 120 240 360

LIFRα

gp130

PY-STAT3STAT3

A

SOCS3

LIF OSM CHX OSM + CHX

0 1 2 4 6 24OSM (hours)

gp130

EtBrStain

DWestern Northern


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P-ERK

0 15 60 120 240 360 0 15 60 120 240 360 0 15 60 120 240 360

gp130

PY-STAT3

STAT3

SOCS3

ERK

OSM OSM + UO126 OSM

STAT3∆

PY-STAT3∆

GFP- (control) GFP+ (STAT3∆)Fig. 4


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Fig. 5A

0 15 60 120 0 15 60 120

LIF Treatment (min)Control UO126

LIFRα

OSMRβ-FLAG

a

0 15 60 120 0 15 60 120

OSM Treatment (min)Control UO126

LIFRα

OSMRβ-FLAG

b

0 15 60 120 0 15 60 120

G-CSF Treatment (min)Control UO126

LIFRα

G-gp130-FLAG

c

PY-STAT3

P-ERK

- + - +d - - + +UO126:LIF:

0 15 60 120Time (min)

0

LIFR

αLe

vel

(% o

f Con

trol)

20

40

60

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120

0 15 60 120 240 360LIF(min)

LIFRα-MYC

0

20

40

60

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120

OSM

Rβ

Leve

l(%

of C

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0 15 60 120 240 360OSM(min)

OSMRβ-FLAG

0

20

40

60

80

100

120G

-gp1

30 L

evel

(% o

f Con

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0 15 60 120 240 360G-CSF(min)

G-gp130-FLAG

LIFIL-6INSPMA

OSM

G-CSF

LIFR

α(S

/A) L

evel

(%

of C

ontro

l)

20

40

60

80

100

120

0 15 60 120 240 360LIF(min)

LIFRα(S/A)-MYC

0

B


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0 15 60 120 0 15 60 120 0 15 60 120 0 15 60 120

Receptor-FLAG

G-CSF Treatment (min)

W.B. Flag

0 15 60 120

(∆cyto)Flag

ND

G-CSF treatment (min)

Fig. 6


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MG - + - - + + OSM - - - + - +

Control OSMRβ

OSMRβ

OSMRβ-Ub-HA 210

210

MG - + - - + + LIF - - - + - +

Control LIFRα

LIFRα

LIFRα-Ub-HA 210

210

MG - - - + OSM - - + -

C gp130

Gp130-Ub-HA

Gp130

210

210

MG - - - + OSM - - + -

C STAT3

STAT3

122

122

Control MG Chloroquine0 120 240 360 0 120 240 360 0 120 240 360

LIFRα

gp130NS

gp130Fragments

CHX(min):A

C a

b

c

d

Control MG Chl. MG+Chl.0 15 60 120 0 15 60 120 0 15 60 120 0 15 60 120

G-gp130-FLAG

G-gp130-FLAG

Fragments

G-CSF(min):B

PY-STAT3

P-ERK

0.1

1

10

100

1000

Control

MGChl.

0.1

1

10

100

0 120 240 360

Control

MG

Chl.

Cycloheximide (min)

Rec

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LIFRα

gp130

a

b

Fig. 7


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Heinz BaumannFrederic Blanchard, Yanping Wang, Erin Kinzie, Laurence Duplomb, Anne Godard and

by distinct mechanismsβ and oncostatin M receptor αreceptor Oncostatin M regulates synthesis and turnover of gp130, leukemia inhibitory factor

published online October 15, 2001J. Biol. Chem.

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The regulation of gp130, leukemia inhibitory factor receptor and