(Received for publication, March 24, 1997)
From the Laboratoire de Biologie Cellulaire, 4 rue Larrey, CHU Angers, 49033 Angers Cedex, France
Oncostatin M (OSM) mediates its bioactivities
through two different heterodimer receptors. They both involve the
gp130-transducing receptor, which dimerizes with either leukemia
inhibitory receptor or with OSM receptor
(OSMR
) to generate,
respectively, type I and type II OSM receptors. Co-precipitation of
gp130-associated proteins, flow cytometry, polymerase chain reaction,
and tyrosine phosphorylation analyses allowed the characterization of
both types of OSM receptors expressed on the surface of different cell lines. It also allowed the detection of a large size protein, p250,
that specifically associates to the type II OSM receptor components and
that is tyrosine-phosphorylated after the activation peak of the
gp130·OSMR
heterocomplex. The restricted expression of type I OSM
receptor by the JAR choriocarcinoma cell line, and type II receptor by
the A375 melanoma cell line, permitted the characterization of their
signaling machineries. Both type I and type II OSM receptors activated
Jak1, Jak2, and Tyk2 receptor-associated tyrosine kinases. The
information is next relayed to the nucleus by the STAT3 transcriptional
activator, which is recruited by both types of OSM receptors. In
addition, STAT5b was specifically activated through the gp130·OSMR
type II heterocomplex.
The signaling pathway differences observed between the common type I LIF/OSM receptor and the specific type II OSM receptor might explain some of the bioactivities specifically displayed by OSM.
Oncostatin M (OSM)1 is a multifunctional cytokine belonging to the interleukin-6 (IL-6) family and that shares many properties with those reported for LIF (1). Both OSM and LIF are able to inhibit the spontaneous differentiation of embryonic stem cells (2). They also induce the terminal differentiation of the M1 murine myeloid cell line (3). Like the other IL-6 family members, OSM has been shown to induce acute phase protein synthesis in hepatocytes (4). Beside these LIF-shared bioactivities, OSM displays some specific properties and can inhibit the growth of a variety of solid tumor cells (5) and triggers in vitro the proliferation of Kaposi's sarcoma-derived cell lines (6, 7). In addition, expression of an oncostatin M transgene in the early T cell lineage stimulates a dramatic accumulation of T cells in the mice lymph nodes (8).
The redundancy of OSM and LIF biological properties is in part
explained by the shared use of a common heterocomplex receptor composed
of the gp130 signal transducing protein associated with the LIF
receptor (LIFR
) component (9, 10). Binding experiments have
pointed out the existence of a second and different high affinity
receptor for OSM (also referred to as type II OSM receptor) (9-11).
Type II OSM receptor complex binds OSM in a specific manner and is not
recognized by LIF (9-11). Type II receptor also involves a
gp130-transducing component that associates with a second receptor subunit very recently isolated as OSM receptor
(OSMR
), which displays an apparent molecular mass of 180 kDa (12). Comparison of
OSMR
to gp130 and LIFR
shows high homology levels in both domain
structure and primary amino acid sequence.
Signaling mediated through the common type I LIF/OSM receptor was studied in detail previously (13-17). Activation of type I receptor by LIF leads to the recruitment of Jak1, Jak2, and Tyk2 receptor-associated kinases, which are in turn relayed by STAT3 transcription factor to transduce the signal to the nucleus.
Until recently the nature of the type II OSM receptor was less well characterized, and very little information regarding its signaling machinery has been reported. It has been shown that the type II OSM receptor recruits the serine-threonine MAP kinase pathway (11), as well as the src-related kinase p62yes (18).
In the present study we analyzed the expression of both type OSM receptors in a series of cell lines, and by using the A375 melanoma cell line specifically expressing the type II OSM receptor complex, we studied its signaling transduction pathway.
A375 melanoma, JAR choriocarcinoma, KB
epidermoid carcinoma, HepG2 hepatoma, and SK-N-MC neuroblastoma cell
lines were obtained from the American Type Culture Collection
(Rockville, MD) and grown in RPMI 1640 culture medium supplemented with
10% fetal calf serum. OSM (2 × 106 units/mg) was
purchased from Peprotech (Canton, MA), and purified recombinant LIF
(108 units/mg) produced in the Chinese hamster ovary cell
line was kindly provided by Dr. K. Turner (Genetics Institute, Boston, MA). B-T6 (IgG1), B-P4 (IgG1), B-K5 (IgG1), and B-R3 (IgG2a) anti-gp130 mAbs were described in detail elsewhere (19). mAb 32209 (IgG2b) directed against LIFR was obtained from R & D Systems (Minneapolis, MN). mAbs recognizing STAT1 (IgG1) and STAT3 (IgG1) were obtained from
Transduction Laboratories (Lexington, KY). Rabbit anti-STAT4, anti-STAT5b, and antibodies recognizing the carboxyl-terminal sequence
of LIFR
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Polyclonal rabbit antibodies directed against Jak1, Jak2, and Tyk2
and 4G10 anti-phosphotyrosine mAb were from Upstate Biotechnology, Inc.
(Lake Placid, NY).
The immunofluorescence studies were performed following the standard protocols. The samples and the appropriate isotype controls were then analyzed on a FACSCAN from Becton Dickinson (Mountain View, CA).
OSMRcDNA was synthesized from 5 µg
of total RNA by using an antisense oligonucleotide located at positions
2050-2073 of OSMR sequence as primer (12). For the PCR analysis 40 amplification cycles (93 °C, 30 s; 57 °C, 1 min; 72 °C, 1 min) were carried out by using a second derived primer corresponding to
the positions 1586-1609 of the OSMR
published cDNA. Amplified
products were analyzed on a 2% agarose gel.
Tyrosine phosphorylation analysis was performed as described in detail previously (19, 20, 23). After immunoprecipitation the complexes were isolated with beads coupled to protein A, subjected to SDS-PAGE, and transferred onto an Immobilon membrane (Millipore, Bedford, MA). The membranes were subsequently incubated with the relevant primary antibody before being incubated with the appropriate second antibody labeled with peroxidase for 60 min. The reaction was visualized on an x-ray film by using the ECL reagent (Amersham Corp., Les Ulis, France) according to the manufacturer's instructions. The membranes were then stripped in 0.1 M glycine HCl, pH 2.5, for 1-6 h depending on the previously used antibodies, neutralized in 1 M Tris-HCl, pH 7.6. Before reblotting, disappearance of all remaining signal was checked by a 30-min film exposure. For cell surface biotinylation, the A375 cells were starved for a 4-6-h period before being stimulated with 50 ng/ml OSM for 5 min. Then, the cells were washed with PBS and incubated for 20 additional minutes in PBS, 0.1 M Hepes, pH 8, 0.5 mg/ml water-soluble sulfo-NHS-biotin (Pierce) (21). After the contact period the reaction was stopped by adding 0.1 M Tris/HCl, pH 8. The cells were lysed and the proteins immunoprecipitated with the B-T6 anti-gp130 mAb and analyzed as described above. After tyrosine phosphorylation analysis and stripping of the blot, the visualization of the biotinylated proteins was carried out by using a streptavidin/peroxidase solution (1/5,000) from Dako (Trappes, France)
DNA-binding ProteinAfter stimulation with OSM, whole cell
extracts were prepared as described previously (24, 25). Solubilized
proteins were incubated for 18 h in the presence of 1 µg of
double strand 5-biotinylated oligonucleotides bound to
streptavidin-agarose beads. STAT3 and STAT5b high affinity interacting
GAS motifs were derived, respectively, from the c-fos gene
(sis-inducible element, SIEM 67)
(5
-CATTTCCCGTAAATCTTGTCG-3
) and from the Fc
R gene (GRR)
(5
-GTATTTCCCAGAAAAGGAAC-3
) as reported before (24, 25). The complexes
were precipitated, subjected to SDS-PAGE, and transferred onto an
Immobilon membrane (Millipore). The membranes were then stained by
using an anti-STAT3 mAb (Transduction Laboratories) or an anti-STAT5
polyclonal antibody (Santa Cruz Biotechnology). Immunoreactive bands
were visualized with ECL Western blotting reagent as described above.
Specificity of the observed signals was controlled by introducing in
OSM-activated cell extracts a 100-fold excess of unlabeled double
strand DNA as competitor for the biotinylated oligonucleotides.
In the present work we analyzed the cell surface
expression of both gp130 and LIFR by flow cytometry in different
cell backgrounds. The obtained results and their correlation with the
sensitivity of the studied cell lines to the OSM cytolytic activity led
us to concentrate our investigation on the A375 melanoma and JAR choriocarcinoma cell lines. Both cell lines expressed the common signaling receptor protein, gp130 (Fig. 1A).
In contrast the LIFR
subunit was only detected on the JAR cell
surface, but not on the A375 melanoma cell line. A PCR analysis
revealed the presence of a very low level of LIFR
gene transcription
in A375 cells, but we could not surface-detect protein (Fig. 1,
A-C). Analysis of the proliferative responses of the two
studied cell lines grown in the presence of OSM showed a strong
inhibition of the A375 cell line growth, but not of the JAR
choriocarcinoma cells. LIF was without effect in both cultures (Ref. 19
and data not shown). The conclusions of these results were similar to
those reported previously by other groups using different approaches
(9, 11, 12, 22). The results indicate that despite the fact of a lack of detectable expression of LIFR
on the A375 cell surface, OSM could
mediate a functional response in this cell line.
We recently characterized the B-T6 anti-gp130 mAb (19). B-T6 is a mute
antibody, which did not interfere with the biological responses
mediated by the cytokines of the IL-6 family, but allows the
co-precipitation of receptor subunits that associate with gp130.
Receptor tyrosine phosphorylation events observed in response to OSM in
A375 and JAR cell lines were studied. Treatment of the JAR cells with
OSM for 10 min resulted in the induction of gp130 tyrosine
phosphorylation and of an additional protein with a molecular mass of
190-210 kDa, co-precipitating with gp130 (Fig. 1B).
Reblotting the filter with an antibody directed against LIFR
identified the associated protein as LIFR
, in agreement with the
FACS patterns.
A similar approach with the A375 cell line allowed the detection of the
activated form of gp130 associated with a protein displaying a slightly
higher molecular mass of 150-180 kDa (Fig. 1B). Membrane
reprobing indicated that the LIFR subunit was not detectable in the
A375 cell line and that the observed 150-180-kDa product very likely
corresponded to the recently cloned gp180/OSMR
(12). This notion was
further reinforced by analyzing the presence of OSMR
mRNA in
A375 and JAR cell lines by PCR amplification (Fig. 1C). The
presence of an OSMR
transcript was clearly detectable in A375
melanoma cells, but not in JAR choriocarcinoma-derived cell line. The
transmembrane expression of OSMR
was studied by labeling the A375
extracellular surface with a water-soluble biotin ester. After
activating the cells with OSM we analyzed the gp130-associated protein(s) that were both tyrosine-phosphorylated and biotinylated (21). The obtained results show the presence in the A375 cells of a
150-180-kDa transmembrane protein very likely corresponding to OSMR
(Fig. 1D). Interestingly, in the absence of activation, a
nonphosphorylated form of gp180 could be to some extent co-precipitated with gp130, as detected after streptavidin/peroxidase staining of the
membrane. A slight decrease in gp130 electrophoresis mobility was also
observed upon OSM activation as noticed previously (23). Treatment of
the A375 cells with LIF did not allow the recruitment of either gp130
or gp180/OSMR
, further reinforcing the specificity of the receptor
expressed by this cell line (Fig. 1B). Moreover, pretreatment of the A375 cells with a molar excess of B-R3 anti-gp130 mAb, previously reported to inhibit the homo- or heterodimerization of
gp130 with its neighboring receptors, abrogated the induction of
tyrosine phosphorylation of both receptor subunits (Ref. 19 and data
not shown). Altogether, our results corroborate results obtained by
performing binding analyses of LIF and OSM and demonstrate a specific
expression of type II OSM receptor at the cell surface of the A375
melanoma cell line (9, 11). In contrast, the JAR choriocarcinoma cell
line only expressed on its surface the common LIF·OSM receptor
complex.
A detailed kinetic study of OSM responses in A375 cells
revealed a simultaneous activation of gp130 and gp180 that was
maximally recruited after a 5-10-min contact with OSM. JAR cell line
was incubated for 10 min with the cytokine and used as control (Fig. 2). Interestingly, in addition to gp130 and OSMR, a
third product with an apparent molecular mass of 250 kDa was detected
in the A375 cell line. Compared with the receptor heterodimer, tyrosine phosphorylation of the 250-kDa protein, or p250, is delayed 10-15 min
and also becomes dephosphorylated after 30 min. Staining of the
membrane with an anti-LIFR
-specific antibody failed to detect any
signal in the A375 cells, but gave a readily detectable band in the JAR
cell line, indicating that p250 was not related to LIFR
. p250 was
not detectable after activation of type I OSM receptor regardless of
the contact time with the activating cytokine (Fig. 2, right
lane, and data not shown). Expression of p250 was not restricted
to the A375 cell line as summarized in Table I, and it
could be co-precipitated with gp130 in all the cell lines expressing
OSMR
/gp180 we have analyzed so far. In contrast, we could not detect
the p250 receptor-associated protein in cell lines expressing solely
the type I OSM receptor. Similarly, by using the SK-N-MC neuroblastoma
cell line that expresses all the known high affinity receptors for the
IL-6 family members (19, 23) (with the exception of OSMR
), we were
not able to co-associate p250 to gp130 after activating the cells by
any of the six related cytokines belonging to this family. This result
suggests a specific recruitment of p250 in the context of the type II
OSM receptor or a lack of p250 expression in the SK-N-MC cells. In line
with our observation, it should be noted that a similar association of
a 250-300-kDa large size protein to type II OSM receptor has already
been observed in some instances (18). These results indicate that
activation of the gp130·gp180 heterocomplex by OSM leads to a delayed
activation of a p250 protein, which seems to interact in a specific way
with the OSM receptor type II complex.
|
Receptor activation of the IL-6/LIF family of cytokines
results in immediate phosphorylation of the transduction subunits by
the Jak family members. Homo- or heterodimerization of the gp130 signal
transducer was shown to induce activation and recruitment of Jak1,
Jak2, and Tyk2 (13-15). It was demonstrated previously that
gp130/LIFR type I OSM receptor utilize the same Jak kinases for
tyrosine phosphorylation (13-15). This led us to investigate whether
the Jak-Tyk kinases were involved in signaling initiated through type
II OSM receptor. Fig. 3A shows that tyrosine
phosphorylation of Jak1, Jak2, and Tyk2 signaling proteins is induced
after treatment of A375 cells by OSM. Tyk2 phosphorylation observed in
response to OSM is weaker than the signals detected for Jak1 and Jak2
as reported previously for the other members of the IL-6 family (13). Nevertheless, immunoblots of lysates revealed a clear expression of
Tyk2. Thus, these data indicate that the Jak signaling cascade induced
by activation of type II OSM receptor is similar to that induced
through the type I OSM receptor as it appears for the JAR cell line
(Fig. 3A).
Downstream signaling events were further analyzed by studying the
transcription factors known as STATs, which are tyrosine-phosphorylated at the cytoplasmic level before being translocated to the nucleus (16,
17). As shown in Fig. 3B, stimulation of the A375 cells with
OSM elicits tyrosine phosphorylation of STAT3 and STAT5b proteins.
Similarly, STAT3 was also recruited by the gp130·LIFR heterocomplex, but STAT5b activation level remained unchanged after OSM
stimulation of the JAR cell line, sustaining the notion of a specific
recruitment of STAT5b by type II OSM receptor. We also looked for a
possible induction of STAT1 and STAT4 tyrosine phosphorylation by
activating both type receptors with OSM, but no detectable signal could
be observed (Fig. 3B and data not shown). To further
establish the activation and recruitment of STAT3 and STAT5b in
response to OSM, interaction of STAT transcription factors with DNA GAS
motifs was studied. DNA target sequences were chosen, respectively, in
the promoters of c-fos and Fc
receptor genes, as
described previously (24, 25). The results presented in Fig.
4 show that activation of type II OSM receptor by its
cognate ligand induces a binding of both STAT3 and STAT5b to DNA.
Several isoforms, or phosphoforms of STAT proteins, were observed in
agreement with the published studies (25). In contrast, and despite a clear expression of STAT5b in the JAR cell line (data not shown), its
activation was not observed in response to the stimulation of type I
LIF/OSM receptor. The obtained results are in line with the tyrosine
phosphorylation study presented in Fig. 3. Altogether these results
indicate that OSM type I and II receptors are both able to activate
STAT3, whereas STAT5b is specifically recruited by the OSM type II
receptor.
Concluding Remarks
Type I OSM receptor involving
gp130/LIFR preferentially recruits the Jak1, Jak2, and Tyk2 tyrosine
kinases as it was reported after its activation by LIF (13). The
information is next relayed to the nucleus by STAT3 transcriptional
activator (16-17). No variation could be observed in the detection and
activation of the STAT1 signaling protein. STAT1 gene inactivation in
the mouse has underlined its essential implication in mediating the
antiviral properties of interferons (26, 27). No evidence for an
alteration of the physiological responses dependent upon the IL-6
cytokine family was detected in the STAT1 deficient mice.
It was demonstrated by Thoma et al. (11) that type II OSM
receptor strongly activates the MAP kinase pathway. In the present work
we observed that the gp130·gp180 heterocomplex was able in addition
to STAT3 to also recruit the STAT5b transcription factor. Similarly, it
was recently demonstrated that IL-6 can activate STAT5b in rat liver
(28). We also pointed out the presence of a large size protein, p250,
which can be co-precipitated with the activated gp130/gp180 subunits,
but whose tyrosine phosphorylation is delayed when compared with the
heterocomplex receptor. Preliminary experiments performed after an
external biotin labeling of the A375 cell surface indicate that p250 is
a transmembrane protein, but we do not know yet whether it can directly
bind to the ligand. It was reported recently that activation of the
-interferon receptor complex could be modulated by a large size
receptor-associated protein that was identified to be the CD45
phosphatase (29). We cannot exclude the eventuality that related
processes might occur to regulate the activation of some of the IL-6
family receptors. Similarly, it was reported that the phosphotyrosine
phosphatase 1D might directly couple the intracytoplasmic portion of
gp130 to activation of the gp130·LIFR
heterocomplex (16).
OSM displays some specific biological properties not shared by LIF and can, for instance, inhibit the growth of solid tumor-derived cell lines (5) and trigger the proliferation of Kaposi's sarcoma-derived cell lines (6, 7) and of some T cells subsets (8). These observed differences might be explained by the tissue distribution of type II OSM receptor and by the specificity of its signaling.
We thank Josy Froger for technical support and Jean-Paul Gislard for assistance with figures.