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*
Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Germany; and
Department of Medical Protein Chemistry, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chains were generated, allowing the
induced heterodimerization of two different cytoplasmic tails. Our
studies demonstrate that upon heterodimerization with the gp130
cytoplasmic region, the cytoplasmic parts of both LIFR and OSMR were
critical for activation of an acute phase protein promoter in HepG2
hepatoma cells. The membrane-proximal region of LIFR or OSMR was
crucial for the ability of such receptor complexes to induce DNA
binding of STAT1 and STAT3 in COS-7 cells. Membrane-distal regions of
LIFR and OSMR contributed to STAT activation even in the absence of
gp130 STAT recruitment sites. We further show that the Janus kinases
Jak1 and Jak2 constitutively associated with receptor constructs
containing the cytoplasmic part of LIFR, OSMR, or gp130, respectively.
Homodimers of the LIFR or OSMR cytoplasmic regions did not elicit
responses in COS-7 cells but did in HepG2 cells and in MCF-7 breast
carcinoma cells. Thus, in spite of extensive functional similarities,
differential signaling abilities of gp130, LIFR, and OSMR may become
evident in a cell-type-specific manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-6-type cytokines have in part overlapping functions: e.g.,
IL-6, LIF, as well as OSM are able to induce the synthesis of acute
phase proteins in hepatocytes (4, 5, 6, 7). All three cytokines
induce macrophage differentiation of mouse promyelocytic M1 cells
(8, 9, 10). This functional redundancy can be explained by
the shared use of the receptor subunit gp130. Whereas IL-6 and IL-11
induce homodimerization of gp130, the other IL-6-type cytokines
lead to heterodimerization of gp130 with the LIF receptor (LIFR) or the
OSM-specific receptor (OSMR) (1, 2). Human, but not
murine, OSM can signal via a LIFR/gp130 heterodimer in addition to the
OSMR/gp130 receptor complex (11, 12). Apart from exerting
these redundant effects, each cytokine is additionally endowed with
specific functions: e.g., LIF plays an important role for blastocyst
implantation (13) and in activation of the
hypothalamic-pituitary-adrenal axis during stress and inflammation
(14). OSM, secreted predominantly by activated macrophages
and T cells, is also produced by AIDS-associated Kaposi sarcoma cells
and mediates their proliferation (15, 16). Thus, the
restricted pattern of cytokine expression and the distribution of the
ligand binding receptors may explain cytokine-specific effects.
Moreover, differences in signaling of homo- vs heterodimers have been
noted: e.g., overexpression of the transcription factor SCL inhibits
the LIF- and OSM-, but not the IL-6-mediated induction of M1 cell
differentiation (17). Dexamethasone inhibits the induction
of the thiostatin gene by LIF but not by IL-6 (18). The
molecular basis underlying these differential signaling events is
currently unknown.
To analyze OSMR and LIFR functions in transfected cells independently of endogenous receptors, various groups have taken advantage of chimeric receptor systems: G-CSF-induced homodimerization of the cytoplasmic region of the OSMR and mutants thereof led to STAT activation and gene induction in hepatoma cells. Some differences to the signal transduction of a gp130/OSMR heterodimer were noted (19). Similarly, ligand-induced homodimerization of the cytoplasmic regions of LIFR led to biological responses in some (19, 20, 21, 22, 23) but not all cells (24, 25), in spite of the fact that the LIFR is only known to signal in combination with gp130.
The aim of this study was to investigate the contribution of the
LIFR and OSMR to signal transduction in heterodimeric complexes with
gp130. We therefore used a receptor system based on the extracellular
parts of the IL-5R - and ß-chains (26) which allows
the directed formation of heterodimers, thereby mimicking the proposed
natural receptor complexes (gp130/LIFR or gp130/OSMR).
Using this system, we demonstrate that the membrane-proximal regions of LIFR or OSMR are crucial for signal transduction in the heterodimeric receptor complex. Only one cytoplasmic tail has to contain STAT recruitment sites and these can be contributed by either gp130, LIFR, or OSMR. We further show constitutive association of Janus kinases not only with gp130, but also with the LIFR and OSMR. Moreover, evidence is provided for distinct signaling characteristics of gp130, LIFR, or OSMR apart from their functional homologies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes and T4-DNA ligase were obtained from
Boehringer Mannheim (Mannheim, Germany) or AGS (Heidelberg, Germany),
and protease inhibitors were obtained from Sigma (Munich, Germany).
DMEM and DMEM/F12 were purchased from Life Technologies (Eggenstein,
Germany) and FCS from Seromed (Berlin, Germany). Human IL-5 was
expressed in Sf9 insect cells and purified as described previously
(27). Human recombinant erythropoietin was kindly provided
by Drs. J. Burg and K.-H. Sellinger (Boehringer Mannheim,
Penzberg, Germany). For flow cytometry, the mAb 16-4 specific for the
human IL-5R -chain (J. Van der Heyden and J. Tavernier, unpublished
results) and the mAb S-16 specific for the human ßc-chain (Santa Cruz
Biotechnology, Santa Cruz, CA) were used. Polyclonal antisera against
human ßc and Jak2 were purchased from Santa Cruz Biotechnology, and
Tyk2-specific antiserum was purchased from Transduction Laboratories
(Lexington, KY). Antiserum against Jak1 was a kind gift from Dr. A.
Ziemicki (Bern, Switzerland). PE-labeled goat anti-mouse
IgG-F(ab')2 was obtained from Dianova (Hamburg,
Germany).
Plasmid construction
The construction of the chimeras IL-5R/gp130, IL-5R/gp130-B1/2,
IL-5Rß/
cyt, IL-5Rß/gp130box1, IL-5R/LIFR, and erythropoietin
receptor (EpoR)/gp130 have been described in previous studies
(26, 28, 29, 30). The mutant IL-5Rß/LIFR-B1/2 was cloned by
deleting a CelII/BamHI-fragment from the
expression plasmid pSVL-IL-5R/LIFR and exchanging the sequence
encoding the extracellular part of IL-5R by the sequence encoding
the corresponding fragment of IL-5Rß. Because of the cloning
procedure, three additional amino acids (Ile-Glu-Thr) were added after
position Glu931, followed by the termination
codon. The IL-5R/OSMR constructs were generated by RT-PCR using a
RT-PCR kit from Boehringer Mannheim. The sense primer annealing
upstream of the sequence encoding the OSMR transmembrane region
contained an in-frame EcoRI site at its 5' end. Thus, the
OSMR sequence starting with Thr727 is preceded by
a phenylalanine residue. The antisense primer contained a
BamHI site next to the stop codon. Total RNA (1 µg)
isolated from OSM-sensitive human A375 melanoma cells was used for RT.
The resulting cDNA was further amplified by PCR using the same primers
as before according to the manufacturer’s instructions. The resulting
OSMR fragment was inserted into EcoRI/BamHI
digested pSVL-based expression vectors for IL-5R/gp130 and
IL-5Rß/gp130, respectively. A series of chimeric receptors encoding
truncated cytoplasmic OSMR domains was generated by PCR using 3'
oligonucleotides incorporating in-frame termination codons followed by
the recognition site for BamHI. OSMR1, OSMR2,
OSMR3, and OSMR-B1/2 retain 191, 162, 153, and 65 amino acids of the
OSMR cytoplasmic tail, respectively. The resulting PCR products were
inserted into the EcoRI- and BamHI-digested
expression plasmid pSVL-IL5Rß/OSMR. The integrity of all constructs
was verified by DNA sequence analyses using an ABI PRISM 310 Genetic
Analyzer (Perkin-Elmer, Norwalk, CT). EpoR/LIFR and EpoR/OSMR
constructs were generated by exchanging the
EcoRI/BamHI fragment of pSVL-EpoR/gp130 encoding
the transmembrane and intracellular region of gp130 by fragments
encoding the corresponding regions of LIFR and OSMR. For transfection
of HepG2 and MCF-7 cells, XhoI/BamHI fragments
comprising the cDNA encoding the various receptor constructs were
cloned into expression vector pCAGGS (31) digested with
XhoI and BglII. Expression plasmids pRK5-Jak1 and
pRK5-Jak2 were kindly provided by Dr. I. Kerr (London, England) and Dr.
J. N. Ihle (Memphis, TN). An expression vector for Tyk2 was
generated by inserting Tyk2 cDNA (generously provided by Dr. S.
Pellegrini, Paris, France) into vector pSVL (Pharmacia, Piscataway,
NJ). The STAT3 expression vector has been described
(30).
Cell culture and transient transfections
Simian monkey kidney cells (COS-7) and the human breast carcinoma cell line MCF-7 were maintained in DMEM, human hepatoma cells (HepG2) in DMEM/F12 medium supplemented with 10% FCS, 100 mg/L streptomycin, and 60 mg/L penicillin. Approximately 1.5 x 107 COS-7 cells were transiently transfected with 10–20 µg plasmid DNA using the DEAE-dextrane method. Briefly, cells were incubated for 90 min in 7.5 ml FCS-free medium containing the plasmid DNA, 6 µl chloroquine (100 mM), and 60 µl DEAE-dextrane (50 mg/ml) for 90 min, avoiding gas exchange. Afterward, cells were incubated for 1 min in PBS containing 10% DMSO. After extensive washing and cultivation for additional 48–72 h, cells were harvested.
HepG2 and MCF-7 cells were transfected with 20 µg plasmid DNA using the calcium-phosphate method as described previously (32).
Flow cytometry
COS-7 cells were released from the dishes using PBS/10 mM EDTA,
washed, and resuspended in cold PBS supplemented with 5% FCS and 0.1%
sodium azide (PBS/azide). Cells (5 x
105–1 x 106) were
incubated with 1 µg/ml of either the monoclonal anti-IL-5R Ab
(16-4) or anti-IL-5Rß Ab (S-16) for 30 min at 4°C. After
washing the cells with cold PBS/azide, they were incubated with a 1/100
dilution of PE-conjugated goat anti-mouse
IgG-F(ab')2 (Dianova) for 30 min at 4°C. Cells
were washed again with cold PBS/azide, resuspended in PBS/azide, and
analyzed by flow cytometry using a FACScalibur (Becton Dickinson,
Mountain View, CA) equipped with a 488-nm argon laser.
EMSA
Forty-eight to 72 h after transfection, COS-7 cells were
starved for 4–6 h and stimulated with 80 ng IL-5/ml for 30 min or with
7 U Epo/ml for 15 min. Nuclear extracts were prepared as described
(33). Protein concentrations were measured with the
Bio-Rad protein assay (Bio-Rad, Richmond, CA). A double-stranded
mutated SIE oligonucleotide from the c-fos promoter (m67SIE:
5'-GAT CCG GGA GGG ATT TAC GGG AAA TGC TG-3') was labeled by filling in
5' protruding ends with the Klenow enzyme using
[-32P]dATP (3000 Ci/mmol; 10 mCi/ml).
Nuclear extracts containing 5–10 µg protein were incubated with
about 10 fmol (10,000 cpm) of probe in gel shift incubation buffer (10
mM HEPES (pH 7.8), 1 mM EDTA, 5 mM MgCl2, 10%
glycerol, 5 µM DTT, 0.7 µM PMSF, 0.1 mg/ml of poly(dI-dC), and 1
mg/ml BSA) for 10 min at room temperature. The protein-DNA complexes
were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol
in 0.25-fold TBE (200 mM Tris, 166 mM boric acid, 2 mM EDTA, adjusted
to pH 8.3) at 20 V/cm for 4 h. Gels were fixed in a water solution
of 10% methanol and 10% acetic acid for 30 min, dried, and
autoradiographed. Data were further analyzed with a Storm 840
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Immunoprecipitations and Western blotting
Forty-eight to 72 h after transfection, COS-7 cells were washed twice with PBS, scraped off the dish, and lysed in BRIJ96-lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% BRIJ96, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin) for 30 min on ice. Cell lysates were centrifuged at 14,000 rpm for 10 min. The supernatants were used for immunoprecipitation of the receptor chimeras using the anti-IL-5Rß Ab S-16. After overnight incubation at 4°C, immune complexes were collected on protein A-Sepharose during a 60-min incubation, washed twice with washing buffer (as lysis buffer, but with only 0.1% BRIJ96), and boiled for 5 min in Laemmli buffer at 95°C. The proteins were separated by 7.5% SDS-PAGE, followed by electroblotting onto a polyvinylidene difluoride membrane (PALL, Dreieich, Germany). Western blot analysis was conducted with the indicated Abs and the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions.
Reporter gene assays
pGL32M-215Luc contains the promoter
region -215 to +8 of the rat 2-macroglobulin
gene upstream of the luciferase-encoding sequence of plasmid pGL3
(Promega, Madison, WI) (32). For reporter gene assays,
HepG2 or MCF-7 cells were transfected with 8 µg of luciferase
reporter construct, 4 µg of ß-galactosidase control plasmid pCH110
(Pharmacia), and 4 µg of each receptor expression vector. Twenty-four
hours after transfection, cultures were subdivided, and after another
12-h recovery period, treated for 24 h with 80 ng/ml IL-5.
Luciferase assays were performed using the Promega luciferase assay
system. The values in each experimental series were normalized to
ß-galactosidase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the contribution of the LIFR and OSMR to signal
transduction in the respective heterodimeric complexes with gp130, we
generated chimeric receptor constructs as shown in Fig. 1
. They consist of the extracellular
domains of IL-5R, IL-5Rß, or the EpoR, and the transmembrane and
intracellular parts of gp130, LIFR, or OSMR. Previous studies have
shown that IL-5 binding affinities of the IL-5R chimeras were not
influenced by deletion or exchange of the cytoplasmic regions
(26). No signaling was observed when only a single
chimera, /gp130 or ß/gp130, was expressed, demonstrating that
signaling-competent receptor complexes have to contain both the -
and the ß-chain chimeras (26). Therefore, this system is
suitable to delineate the relative contributions of LIFR or OSMR to
signal transduction within a heterodimer with gp130.
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/gp130. After transient transfection and
IL-5 stimulation of both receptor combinations (/gp130 + ß/LIFR,
/gp130 + ß/OSMR), a strong induction of a luciferase reporter gene
under the control of the 2-macroglobulin
promoter was detectable (Fig. 2). This
induction was comparable to that observed upon induced homodimerization
of gp130 cytoplasmic tails (/gp130 + ß/gp130). /gp130 did not
elicit luciferase expression when dimerized with ß/cyt, a receptor
chimera devoid of a cytoplasmic region (Fig. 2). This indicates that
the cytoplasmic parts of LIFR and OSMR crucially contribute to
signaling in heterodimeric complexes with /gp130.
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To achieve higher transfection efficiencies, we switched to COS-7
cells which enabled us to monitor surface expression of receptor
chimeras by flow cytometry and to measure DNA binding of STATs by EMSA.
When cells expressing /gp130 + ß/gp130 were stimulated with IL-5,
STAT1 became activated as revealed by the appearance of a respective
band in gel shift assays (Fig. 3A, left panel; see
Ref. 26). However, the STAT DNA binding activity induced
by heterodimerization of the cytoplasmic parts of LIFR and gp130 was
less pronounced relative to the one induced by gp130 homodimerization,
although LIFR and gp130 chimeras were equally well expressed as shown
by FACS analysis (Fig. 3B). DNA binding activity of
endogenous STAT3 is hardly detectable in COS-7 cells (Fig. 3A, left panel; see Refs. 26, 30), possibly due to a low expression level of STAT3. Upon
overexpression of STAT3, IL-5 stimulation of the various receptor
combinations resulted in a slower migrating protein-DNA complex (Fig. 3A, right panel) which could be supershifted with
anti-STAT3 Abs (data not shown). Also, in the presence of
overexpressed STAT3, both gp130/LIFR heterodimeric receptor complexes
elicited weaker STAT responses compared with the /ß-gp130
homodimer.
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/gp130-B1/2; ß/LIFR-B1/2) that
lack the cytoplasmic regions distal from box1/2. Interestingly, a
receptor combination of /gp130-B1/2 with full-length ß/LIFR was
able to induce a STAT signal, whereas a combination in which only the
membrane-proximal parts of the LIFR and gp130 were present was not
functional (Fig. 4, left
panel). Thus, the C-terminal region of the LIFR can mediate STAT
activation in combination with gp130-B1/2, suggesting that the LIFR
tyrosine motifs serve as STAT recruitment sites. Moreover, also the
membrane-proximal region of the LIFR contributes to signaling in
combination with gp130: whereas a receptor combination in which only
one gp130 cytoplasmic chain was present (/gp130 + ß/cyt) did
not elicit STAT activation, the combination with the membrane-proximal
region of the LIFR (/gp130 + ß/LIFR-B1/2) resulted in a STAT
signal (Fig. 4, middle panel), which was somewhat weaker
than the one elicited by the full-length receptor combination (shown
for comparison in the right panel of Fig. 4).
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). Membrane-proximal and -distal regions of the OSMR contribute to STAT activation in a heterodimer with gp130
We next investigated the signaling potential of OSMR in
combination with gp130 using IL-5R chimeras containing the
transmembrane and cytoplasmic region of OSMR (see Fig. 1). As observed
for the LIFR chimera, activation of STAT1 upon IL-5 stimulation of
heterodimeric OSMR/gp130 chimeras was weaker than the one observed upon
homodimerization of gp130 cytoplasmic tails (Fig. 5A, left panel).
However, STAT3, when overexpressed, was more strongly activated by the
OSMR/gp130 heterodimer (Fig. 5A, right panel),
indicating that the OSMR might be a very potent activator of STAT3.
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1, OSMR2, OSMR3) resulted in
higher expression levels as demonstrated by Western blot analysis (Fig. 5B). However, induced heterodimerization of /gp130 with
truncated ß/OSMR constructs did not lead to increased STAT signals.
Indeed, loss of the C-terminal regions of OSMR resulted in less intense
EMSA bands (Fig. 5C) and reduced
2-macroglobulin promoter activation in HepG2
cells (data not shown) compared with the combination of full-length
receptors. This indicates a function of the OSMR membrane-distal part
for signal transduction which is further demonstrated in Fig. 6 (left panel): a
receptor combination devoid of the gp130 STAT recruitment sites
(/gp130-B1/2 + ß/OSMR) is still able to elicit STAT activation.
Apart from the C-terminal region, membrane-proximal sequences of the
OSMR also play an important role for signal transduction via an
OSMR/gp130 heterodimer: combination of the full-length cytoplasmic part
of gp130 with the OSMR box1/2 region resulted in a strong STAT response
(Fig. 6, right panel). Thus, also the OSMR, as shown for the
LIFR above, provides functional motifs necessary for signaling in
combination with gp130.
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Janus kinases are known to associate with the membrane-proximal region of cytokine receptors. IL-6-type cytokines lead to the activation of Janus kinases Jak1, Jak2, and Tyk2. Therefore, we compared the three receptor subunits gp130, LIFR, and OSMR with respect to their ability to associate with Jaks.
COS-7 cells were cotransfected with expression vectors encoding
ß/gp130, ß/LIFR, or ß/OSMR1 (we used the deletion construct to
achieve higher expression levels, see Fig. 5B) and one of
the three Janus kinases. After lysis under mild conditions, Jak1 and
Jak2 could be coimmunoprecipitated with ß/gp130, ß/LIFR, and
ß/OSMR1 (Fig. 7, upper
panels). Binding of Tyk2 could be shown for ß/gp130 and ß/LIFR
(Fig. 7, lower panel), but not convincingly for ß/OSMR1
(data not shown). Jaks did not bind to a gp130 construct with a
deletion in the membrane-proximal region including the box1 motif (Fig. 7). Moreover, no unspecific precipitation was observed in the absence
of receptor chimeras (data not shown). Western blots of cellular
lysates demonstrate comparable expression levels of each kinase within
one set of experiments. Although the OSMR construct was expressed to a
lower degree (see above), approximately equivalent amounts of receptor
were precipitated, indicating that the anti-IL-5Rß Ab was used in
limiting concentrations (Fig. 7).
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Although LIFR and OSMR are only known to participate in
heterodimeric receptor complexes with gp130, artificial
homodimerization of the respective cytoplasmic regions by chimeric
receptors elicited cellular responses in several studies
(19, 20, 21, 22, 23, 24) but not in others (24, 25). By
combining the respective IL-5R and ß chimeras, we studied the
effects of homodimerization of the cytoplasmic parts of the LIFR or
OSMR. Compared with IL-5R/gp130 homodimers, both the LIFR and the OSMR
homodimers yielded only marginal activation of endogenous STAT1 or
overexpressed STAT3 in COS-7 cells (Fig. 8A). In addition, we replaced
the extracellular region of the IL-5R by the one of the EpoR, a
homodimer, which has been successfully applied for construction of
hybrid receptors (30, 32, 35). However, also the
Epo-induced homodimerization of the cytoplasmic parts of LIFR or OSMR
elicited only marginal STAT responses, whereas homodimerization of
EpoR/gp130 led to a strong signal (Fig. 8B). In striking
contrast, homodimerized cytoplasmic parts of LIFR and OSMR were able to
induce reporter gene activation in HepG2 hepatoma and MCF-7 breast
carcinoma cells (Fig. 8C). Thus, the signaling ability of
LIFR and OSMR homodimers seems to depend on the cellular context.
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C), underlining the functional
homology of these three receptors involved in signaling of all
IL-6-type cytokines. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dimerization of the membrane-proximal region of gp130 with the
full-length cytoplasmic parts of LIFR or OSMR but not with the
respective truncated box1/2 constructs leads to STAT activation (Figs. 4 and 6). This finding demonstrates that 1) the membrane-distal parts
of the LIFR or OSMR provide critical STAT recruitment sites, 2) that
they are functional even in the absence of the gp130 membrane-distal
region, and 3) that only a single chain within such a receptor dimer
has to be equipped with STAT recruitment sites. We show for the first
time that only the membrane-proximal region of gp130 is sufficient to
allow signal transduction when dimerized with full-length LIFR or OSMR
cytoplasmic parts. Their ability to activate STATs in the absence of
gp130 STAT recruitment sites was so far demonstrated only upon
artificially induced homodimerization of the cytoplasmic parts of LIFR
or OSMR (19, 20, 21, 22, 23). The contribution of OSMR STAT
recruitment modules was also implicated by our finding that successive
C-terminal truncations of the OSMR led to decreased STAT responses in
COS-7 cells (Fig. 5B) and
2-macroglobulin promoter induction in HepG2
cells (data not shown) when dimerized with full-length cytoplasmic
gp130. Intriguingly, however, a C-terminal truncation of only 36 amino
acids of the OSMR totally abolished the ability of the OSMR/gp130
complex to activate STATs in Hep3B hepatoma cells (19). In
accordance with the study by Kuropatwinski et al. (19), we
could demonstrate that C-terminal deletions of the OSMR lead to
enhanced protein expression. Future studies will aim at the
identification of the molecular basis underlying this phenomenon.
For the chimeric LIFR/gp130 heterodimer, we observed a lower STAT1- and
STAT3-activating potential in spite of equal expression levels of the
surface receptors (Fig. 3). It is very unlikely that this is due to a
lower affinity for IL-5 since the affinity of the IL-5R does not seem
to be influenced by the receptor’s intracellular region: we have
previously demonstrated that the affinity of IL-5R/gp130 chimeras (such
as the affinity conversion upon coexpression of a IL-5Rß chimera) is
comparable to the one observed for the wild-type IL-5R complex in COS
transfectants (26). Also, receptor complexes incompetent
of signal transduction such as /gp130 + ß/cyt (see Figs. 4 and 6) or IL-5R/gp130 chimeras, fused downstream of the transmembrane
region, bound IL-5 with normal high affinity (Ref. 26 ;
H. M. Hermanns, unpublished data).
Overexpression of STAT3 in COS-7 cells revealed a difference in
signaling via OSMR vs LIFR: coexpression of STAT3 significantly
increased the STAT signal elicited by heterodimeric gp130/OSMR chimeras
relative to that induced by homodimers of the gp130 cytoplasmic part
(Fig. 5), whereas the signal intensity of heterodimeric gp130/LIFR
chimeras remained unchanged (Fig. 3). This suggests that the OSMR,
compared with the LIFR, may be a more potent activator of STAT3,
although it contains only two tyrosine modules corresponding to the
consensus sequence for STAT3 activation YXXQ (30, 38):
Y917 (YVSQ) and Y945 (YKMQ), whereas the LIFR contains three such
motifs: Y981 (YQPQ), Y1001 (YKPQ), and Y1028 (YRPQ).
Cytokines signaling via homomeric LIFR or OSMR complexes (devoid of gp130) are currently unknown. Nonetheless, IL-5 induced homodimerization of the cytoplasmic regions of LIFR and OSMR elicited signals in HepG2 hepatoma and in MCF-7 breast carcinoma cells. In COS-7 cells, however, IL-5R/LIFR or IL-5R/OSMR homodimers, as well as corresponding receptors containing the extracellular part of EpoR, elicited only very weak STAT signals. Several authors have investigated the signaling ability of homodimerized LIFR cytoplasmic chains with partly contradictory results: signaling could be demonstrated in hepatoma and neuroblastoma cells (20), embryonic stem cells (24, 43), and COS-1 cells (21) when chimeric receptors were used that homodimerized upon stimulation with their respective ligands (G-CSF, neurotrophin-3, or epidermal growth factor). On the other hand, G-CSFR/LIFR constructs did not elicit signaling in M1 promyelocytic cells or BAF/03 pre-B cells (24). Similarly, Nakamura et al. (25) did not observe signaling upon GM-CSF-induced homodimerization of two LIFR cytoplasmic parts in embryonic stem cells, although GM-CSF-induced heterodimers of the cytoplasmic parts of LIFR and gp130 elicited a response. However, GM-CSF-induced homodimers of LIFR cytoplasmic regions were able to induce differentiation in M1 and WEHI-3B promyelocytic cells (23). Thus, the cellular background and maybe even the subline used, the transfected hybrid receptors, and the different experimental readouts may affect the results of such studies so that it is difficult to draw general conclusions. It will be of interest to find out to what degree the apparent cell-type specific signaling of chimeric LIFR homodimers might be due to differences in expression of the various Jaks or in their activation profile. The signaling ability of homodimers of the OSMR cytoplasmic region has been addressed so far only in one study (19).
Our study provides evidence that the three signal-transducing
polypeptide chains gp130, LIFR, and OSMR relevant for signaling of
IL-6-type cytokines have common properties: they critically contribute
to signaling via heterodimeric gp130/LIFR or gp130/OSMR receptor
complexes and both membrane-proximal and membrane-distal parts are
involved. Their functional homology could further be demonstrated by
their interchangeability: even enforced heterodimerization of the
cytoplasmic parts of LIFR and OSMR elicited reporter gene activation in
HepG2 hepatoma and MCF-7 breast carcinoma cells (Fig. 8C).
Apart from common functions, the differential signaling ability of
homodimeric receptors in COS-7 cells points at distinct characteristics
of gp130, LIFR, or OSMR, which possibly become evident only in certain
cells. Redundant effects of IL-6-type cytokines have been attributed to
the shared usage of gp130 as a common signal-transducing chain and the
structural as well as functional similarity of gp130, LIFR, and OSMR.
Thus, many of the biological effects elicited by a gp130 homodimer can
be also observed for the LIFR/gp130 or OSMR/gp130 heterodimer, if the
corresponding ligand binding receptor chains are provided. However,
several reports have pointed at differences in signal transduction
events elicited from gp130/gp130 homodimers and gp130/LIFR or
gp130/OSMR heterodimers which cannot be explained by differential
receptor expression (e.g., different preferences in STAT activation
(19, 44, 45), differentiation of M1 transfectants
(17) and PC-12 pheochromocytoma cells (44),
and proliferation vs growth inhibition of breast carcinoma cells
(46, 47, 48)). Therefore, our receptor chimeras are promising
tools to analyze the molecular basis of these differences in signal
transduction via heterodimeric gp130/LIFR and gp130/OSMR complexes.
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Acknowledgments |
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Footnotes |
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2 H.M.H. and S.R. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. I. Behrmann, Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail address:
4 Abbreviations used in this paper: OSM, oncostatin M; OSMR, OSM receptor; LIFR, LIF receptor; Epo, erythropoietin; EpoR, Epo receptor.
Received for publication May 18, 1999. Accepted for publication October 7, 1999.
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- and gp130-mediated stimulation of mitogen-activated protein kinase: evidence for participation of multiple signaling pathways which converge at Ras. J. Biol. Chem. 272:16631.-chain and gp130 in cell type-specific signal transduction. J. Biol. Chem. 272:19982.-chain cytoplasmic domain is sufficient for hemopoietic cell proliferation and differentiation. J. Biol. Chem. 273:34370.This article has been cited by other articles:
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S. Radtke, H. M. Hermanns, C. Haan, H. Schmitz-Van de Leur, H. Gascan, P. C. Heinrich, and I. Behrmann Novel Role of Janus Kinase 1 in the Regulation of Oncostatin M Receptor Surface Expression J. Biol. Chem., March 22, 2002; 277(13): 11297 - 11305. [Abstract] [Full Text] [PDF]
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H. M. Hermanns, S. Radtke, F. Schaper, P. C. Heinrich, and I. Behrmann Non-redundant Signal Transduction of Interleukin-6-type Cytokines. THE ADAPTER PROTEIN Shc IS SPECIFICALLY RECRUITED TO THE ONCOSTATIN M RECEPTOR J. Biol. Chem., December 22, 2000; 275(52): 40742 - 40748. [Abstract] [Full Text] [PDF]
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C. Haan, H. Is'harc, H. M. Hermanns, H. Schmitz-Van de Leur, I. M. Kerr, P. C. Heinrich, J. Grotzinger, and I. Behrmann Mapping of a Region within the N Terminus of Jak1 Involved in Cytokine Receptor Interaction J. Biol. Chem., September 28, 2001; 276(40): 37451 - 37458. [Abstract] [Full Text] [PDF]
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