From INSERM EMI-9928, CHU d'Angers, 4 rue Larrey,
49003 Angers, France and the ** Centre d'Immunologie Pierre Fabre, 5 avenue Napoléon III,
74164 Saint Julien en Genevois, France
Received for publication, February 22, 2001, and in revised form, April 4, 2001
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ABSTRACT |
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Ciliary neurotrophic factor (CNTF) is a cytokine
supporting the differentiation and survival of a number of neural cell
types. Its receptor complex consists of a ligand-binding component,
CNTF receptor (CNTFR), associated with two signaling receptor
components, gp130 and leukemia inhibitory factor receptor (LIFR).
Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor
cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that
displayed activities only on cells expressing the tripartite CNTF
receptor on their surface. In this present study we examined the
membrane binding of the CLC/CLF composite cytokine and observed a
preferential interaction of the cytokine with the CNTFR subunit.
Signaling pathways recruited by the CLC/CLF complex in human
neuroblastoma cell lines were also analyzed in detail. The results
obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2)
leading to a tyrosine phosphorylation of the gp130 and LIFR. The
phosphorylated signaling receptors served in turn as docking proteins
for signal transducing molecules such as STAT3 and SHP-2. In
vitro analysis revealed that the gp130-LIFR pathway could also
stimulate the phosphatidylinositol 3-kinase and the
mitogen-activated protein kinase pathways. In contrast to that reported
before for CNTF, soluble CNTFR failed to promote the action CLC/CLF,
and an absolute requirement of the membrane form of CNTFR was required
to generate a functional response to the composite cytokine. This study
reinforces the functional similarity between CNTF and the CLC/CLF
composite cytokine defining the second ligand for CNTFR.
Ciliary neurotrophic factor was initially named based on its
ability to maintain the survival of parasympathetic neurons of chick
ciliary ganglions (1, 2). Subsequent studies have revealed that
CNTF1 also enhances the
survival of sensory neurons (3), motor neurons (4), and cerebellar and
hyppocampal neurons (5, 6). It can also prevent lesion-induced
degeneration of motor neurons and slows disease progression in mice
with inherited neuromuscular deficits (7-9). The possibility that CNTF
acts as a nerve-derived myotrophic factor has also been established
(10, 11).
CNTF is a member of a family of structurally related cytokines that
includes leukemia inhibitory factor (LIF), interleukin (IL)-6, IL-11,
oncostatin M, cardiotrophin-1 (12-14), and cardiotrophin-like cytokine (CLC) (15, 16). These cytokines share one or both of the
receptor signal transducing subunits gp130 or LIF receptor (LIFR) in
their respective receptor complexes (17-20). The functional CNTF
receptor is a ternary complex that in addition to gp130 and LIFR
receptor also includes a specificity-determining binding component
designed CNTF receptor (CNTFR) anchored to the membrane through a
glycosylphosphatidylinositol motif (21-25). Association of CNTF to the
CNTFR components subsequently leads to gp130-LIFR dimerization and
activation event via the recruitment of Janus kinases (JAK1, JAK2, and
TYK2) (26-30). Tyrosine-phosphorylated gp130 and LIFR in turn serve as
docking proteins for signal transducing molecules such as STAT3 and
SHP-2 (27, 31-35). In vitro studies have also shown that
the gp130-LIFR pathway can stimulate the PI 3-kinase and the MAP kinase
activity (36-39).
The existence of a second ligand for CNTFR was suggested by a study
comparing the phenotypic consequences of disrupting CNTF versus CNTFR genes (40, 41). Unlike mice lacking CNTF, those lacking CNTFR die perinatally and display severe motor neuron deficits.
Thus, the CNTFR subunit plays a critical role during development by
serving as a receptor for a second, developmentally important ligand.
Moreover, a null mutation in the human CNTF gene does not lead to
neurological disease (42). CLC is a recently identified member of the
CNTF/LIF family of cytokines isolated by expressed sequence tag data
base screening and is also referred to as novel neurotrophin-1 or B
cell-stimulating factor-3 (15, 16). We recently showed that CLC
associates with the orphan soluble receptor cytokine-like factor-1
(CLF) (43, 44) to form a composite cytokine (45). CLF expression is
required for CLC secretion, and the CLC/CLF heterocomplex displays
functional activities exclusively on those cells and tissues
co-expressing the tripartite CNTF receptor components. Thus, the
CLC/CLF composite cytokine defines the long sought second ligand for
CNTFR. In the present study we examined the signaling pathways
recruited by the CLC/CLF composite cytokine in human cell lines of
neural origin.
Cells and Reagents--
IMR-32 and SK-N-GP human neuroblastoma
cell lines and the COS-7 cell line (American Type Culture Collection,
Manassas, VA) were routinely grown in RPMI culture medium supplemented
with 10% fetal calf serum. Human CNTF, LIF, IL-2, and the soluble form of CNTFR (sCNTFR) were purchased from R & D Systems (Minneapolis, MN). The 4G10 anti-phosphotyrosine mAb was bought from Upstate Biotechnology Inc. (Lake Placid, NY), and anti-protein C epitope mAb
was from Roche Diagnostics (Meylan, France). Antibodies raised against
STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, SHP-2, JAK1, JAK2, JAK3,
TYK2, p85 (PI 3-kinase), and polyclonal antibodies directed against the
carboxyl-terminal portions of gp130 and LIFR were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Antibodies detecting
phospho-ERK1/ERK2, phospho-STAT3 (Tyr705) and
phospho-AKT were purchased from New England Biolabs (Beverly, MA). The
monoclonal antibodies directed against the human forms of LIFR
(AN-E1, IgG1), gp130 (AN-HH1, IgG2a), CNTFR (AN-C2, IgG1), and
CLF (AN-FC6, IgG1) generated in our laboratory
(44-46) were used for FACS staining.
Receptor immunoprecipitation steps were carried out using AN-G30
(anti-gp130 and IgG1) and AN-E1 (anti-LIFR and IgG1) mAbs.
COS-7 Cell Transfection and cDNAs--
The COS-7 cells used
in this study were intentionally chosen for their low endogenous
expression levels of gp130 and LIFR (47). The cDNAs encoding LIFR,
gp130, and CNTFR were cloned in pCMX expression vector. CLC and CLF
cDNAs were cloned in pcDNA3. The CLC sequence was modified by
introducing a c-Myc epitope (EQKLISEEDL) followed by the protein C
peptide epitope (EDQVDPRLIDGK) at the COOH terminus extremity of the
protein. COS-7 cells were transfected using the DEAE-dextran method as
described previously (47). For each experiment, a control plate was
transfected with a Cytokine Purification--
Cell culture media were concentrated
using Centricon concentrators (Millipore, Bedford, MA) and loaded at
4 °C on an anti-protein C affinity column (Roche). Following
extensive washing (100 mM phosphate, 1 M NaCl,
1 mM CaCl2, pH 7.6), proteins were eluted from
the affinity matrix by calcium ion removal (5 mM EDTA in phosphate-buffered saline) (45). Some of the preparations were further
purified by a quaternary aminoethyl high pressure liquid chromatography step eluted with a NaCl gradient. Protein concentration was determined by SDS-polyacrylamide gel electrophoresis and silver staining using a bovine serum albumin protein standard.
Flow Cytometry Analysis--
Cells were successively incubated
for 30 min at 4 °C with the appropriate primary antibody or isotype
control antibody (10 µg/ml) and a phycoerythrin-conjugated anti-mouse
antibody. Fluorescence was subsequently analyzed on a FACScan flow
cytometer from Becton Dickinson (Mountain View, CA).
Tyrosine Phosphorylation Analysis--
After a 24-h serum
starvation, the cells were stimulated for 10 min in the presence of the
indicated cytokine. The cells were next lysed in 10 mM
Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride,
1 mM sodium orthovanadate, proteinase inhibitors (1 µg/ml
pepstatin, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride), and 1% Nonidet P-40 or Brij 96 depending on the experiments (49). After pelleting insoluble material
and protein standardization, the supernatants were immunoprecipitated
overnight. The complexes were then isolated with beads coupled to
protein A, submitted to SDS-polyacrylamide gel electrophoresis, and
transferred onto an Immobilon membrane (Millipore). The membranes were
subsequently incubated with the relevant primary antibody before being
incubated with the appropriate secondary antibody labeled with
peroxidase for 60 min. The reaction was visualized on an x-ray film
using the ECL reagent (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. In some experiments the membranes were
stripped overnight in 0.1 M glycine-HCl, pH 2.7, and
neutralized in 1 M Tris-HCl, pH 7.6, before reblotting.
Biological Assays--
For proliferation assays, TF1 or
derivatives of Ba/F3 cell lines were seeded in 96-well plates at a
concentration of 5-10 × 103 cells/well in RPMI 1640 medium containing 5% fetal calf serum (46). Serial dilutions of the
cytokines tested were performed in triplicate. After a 72-h incubation
period, 0.5 µCi of [3H]thymidine was added to
each well for the last 4 h of the culture, and the incorporated
radioactivity was determined by scintillation counting. For the KB
transfection experiments, CNTFR cDNA was cloned in the episomal
expression vector pEBS-PL as described previously (45). Transfections
were carried out using the lipid reagent Fugene 6 (Roche), and 48 h later cells were serum-starved and activated for 10 min with
cytokines, as indicated. ERK1 and ERK2 tyrosine phosphorylation levels
were analyzed as described above.
Reporter Gene Activity--
Transient transfection of SK-N-GP
cells was carried out in 24-well culture plates using the Exgen®
transfection reagent (Euromedex, Souffelweyersheim, France). The cells
were transfected with 300 ng of reporter gene as described
previously (50). 48 h after transfection, the cells were incubated
with medium alone, LIF, CNTF, CLC/CLF, or IL-2 for an additional
18 h. Transfected cells were washed twice with ice-cold
phosphate-buffered saline, and 100 µl of lysis buffer was added to
the wells (0.1 M KH2PO4, pH 7.8, 0.1% Triton X-100). The extracts were then used directly to measure
the luciferase activity by integrating total light emission over
10 s using a Packard Topcount luminometer (Meriden, CT).
Luciferase activity was normalized based on protein concentrations.
Binding of CLC/CLF to the Three Components Defining the High
Affinity CNTF Receptor--
To assess the binding capacity of CLC/CLF
to components of the CNTF receptor complex, reconstitution experiments
were carried out using the COS-7 cell line. The three components
defining the high affinity CNTF receptor were expressed alone, by
pairs, or together. 48 h after transfection, the cells were
incubated in the presence of the CLC/CLF heteromeric cytokine tagged
with a c-Myc epitope at CLC carboxyl terminus. Binding to the cell
surface was monitored by flow cytometry using an anti-c-Myc antibody
allowing the recognition of tagged CLC
bound to the cell surface (45) (Table I and Fig.
1). Membrane expression of gp130 or LIFR
or their co-expression did not allow any CLC/CLF binding, indicating that their contribution to CLC/CLF binding is not essential. In contrast, CNTFR expression in COS-7 cells led to the detection of a
strong specific signal, showing a preferential association of the
dimeric cytokine to the latter receptor component. Similar results were
also observed when CNTFR was expressed together with gp130 and LIFR.
Nevertheless, the immunofluorescence peak values were usually 3-fold
higher when the tripartite receptor was expressed compared with the
values observed for CNTFR expressed alone. This probably reflects a
higher and increased affinity of the cytokine for its complete
tripartite receptor. Binding of CLC/CLF to cell surface was also
monitored with an mAb directed against CLF (Table I and Fig. 1). The
results obtained were similar to those observed when using the
anti-c-Myc mAb, indicating that both components remained associated
when they bound to the membrane.
Recruitment of JAK Kinases by CLC/CLF--
Signaling studies were
then carried out using IMR-32 and SK-N-GP human neuroblastoma cell
lines that express on their surface the three subunits defining the
CLC/CLF functional receptor (Fig. 2). We
first analyzed the ability of CLC/CLF complex to bind to the
neuroblastoma cell surface. The results obtained clearly indicated membrane binding of both subunits of the composite cytokine (Fig. 2).
We next analyzed the involvement of JAK kinases in receptor activation
in IMR-32 and SK-N-GP neuroblastoma cell lines (Fig. 3). A 10-min contact of cells with
CLC/CLF led to an activation of JAK1, JAK2, and TYK2, as shown by
analyzing the tyrosine phosphorylation content of these receptor
associated kinases. Nevertheless, despite a clear TYK2 expression in
neuroblastoma cells, its tyrosine phosphorylation induction was usually
weaker than the signals detected for JAK1 and JAK2 in response to
CLC/CLF. A similar situation was reported previously for other members
of IL-6 family (49). No recruitment of JAK3 by CLC/CLF could be
demonstrated (data not shown).
Once activated, JAKs are known to stimulate the phosphorylation of the
gp130 and LIFR subunits of the CNTF receptor on tyrosine residues,
which then serve as docking sites for SH2 containing signaling proteins
(27). We reported previously that binding of CLC/CLF to its tripartite
receptor complex rapidly induced tyrosine phosphorylation of both gp130
and LIFR subunits as well as their heterodimerization in the SK-N-GP
cell line (45). Similarly, both gp130 and LIFR signaling components
were recruited and phosphorylated by the composite cytokine in IMR-32
cells (Fig. 4).
CLC/CLF Activates STAT1 and STAT3--
Downstream signaling events
were further analyzed in IMR-32 and SK-N-GP neuroblastoma cell lines by
studying the activation level of STAT proteins in response to CLC/CLF.
As shown in Fig. 5, cell stimulation with
either LIF, CNTF, or CLC/CLF elicited activation of STAT3 and to a
weaker extent STAT1. Kinetic analysis of STAT3 activation shows that
the observed signal peaked after a 20-min contact prior to decreasing
at 2 h. This is in agreement with the typical behavior of STAT
proteins following their recruitment by the IL-6 type cytokines (31,
32). Tyrosine phosphorylation analyses of the other members of the STAT
family (STAT2, STAT4, STAT5, and STAT6) were carried out, but no
activation could be detected in response to CLC/CLF or to CNTF, as
summarized in Table II.
The transcriptional activity of STAT3 in response to CLC/CLF was then
studied. For this, SK-N-GP cells were transfected with a reporter
construct containing three STAT3 consensus binding sites located
upstream of a thymidine kinase minimal promoter (50). 48 h
post-transfection, the cells were serum-starved and stimulated for an
additional 15 h with saturating amounts of LIF, CNTF, CLC/CLF, or
an irrelevant cytokine (Fig. 6). A
3-4-fold increase of luciferase expression was induced by CLC/CLF, as
well as by LIF and CNTF. Altogether, these data indicate that CLC/CLF recruits STAT3 for both signaling and transcriptional activation of
target genes.
Involvement of SHP-2 and AKT Pathway in CLC/CLF Signaling--
The
tyrosine phosphatase SHP-2 is known to be recruited into the tyrosine
kinase signaling pathway via its binding to phosphotyrosine motifs
expressed by gp130 and LIFR (27). CLC/CLF induced the tyrosine
phosphorylation of SHP-2 as reported previously with CNTF and LIF (27)
(Fig. 7, A and B).
The association of SHP-2 with gp130 was further demonstrated by the
co-immunoprecipitation of gp130 with the phosphatase (Fig.
7C). Similarly, an association between LIFR and SHP-2 was
also seen.
It was reported previously that SHP-2 could regulate gp130 signaling by
recruiting the PI 3-kinase/Akt pathway (51). We therefore analyzed the
interaction between PI 3-kinase and SHP-2. The cell lysates were
incubated with an anti-SHP-2 antibody, and the purified fraction was
analyzed for its content of p85, the regulatory subunit of PI
3-kinase. We observed that the p85 regulatory subunit could be
co-purified with SHP-2 following stimulation of neuroblastoma cells
with CLC/CLF (Fig. 7C).
PI 3-kinase recruitment by CLC/CLF also led to a marked increase in its
tyrosine phosphorylation content, as well as in its association with
AKT (Fig. 8). Comparable results were
obtained when treating the cells with LIF or CNTF in both tested cell
lines. AKT tyrosine phosphorylation was optimal after a 10-min contact with CLC/CLF or CNTF, before to gradually decreasing after 80-100 min
(Fig. 8C).
CLC/CLF Induces the MAP Kinase Pathway Activation--
In addition
to the PI 3-kinase/AKT activation pathway, SHP-2 is also known to
associate with the GRAB2/Sos adaptators and regulate the MAP kinase
pathway (36, 52, 53). ERK1 and ERK2 involved in the MAP kinase pathway
have been shown to play important roles in mediating the mitogenic
effects of the IL-6 family members. ERK1 and ERK2 activation was
determined by measuring their tyrosine phosphorylation levels.
Stimulation of the neuroblastoma cell lines with CLC/CLF quickly
increased basal values (Fig. 9).
Interestingly, the activation was quick and transient. The values went
back to basal levels after a 20-30-min contact. These results
demonstrate involvement of the MAP kinase signaling pathway in
functional responses to the CLC/CLF composite cytokine.
Soluble CNTFR Failed to Promote the CLC/CLF Response--
In a
collaborative study we reported previously that cell lines not normally
responsive to CNTF responded to treatment with a combination of CNTF
and the sCNTFR component (23). Additionally, we also observed that
CLC/CLF could, in vitro or on the cell surface, directly
contact sCNTFR (Ref. 45 and Fig. 1). Therefore, experiments were
carried out using the TF1 erythroleukemia cell line (23), and
derivatives of the IL-3-dependent Ba/F3 cell line were
rendered responsive to LIF or to CNTF by transfection with the
appropriate receptor chains (Ref. 46 and Fig.
10). Both TF1 and BA/F3 cell lines
expressing gp130 and LIFR responded to LIF and to a combination of CNTF
and sCNTFR, as reported before (Ref. 23 and Fig. 10, A and
B). Interestingly, sCNTFR entirely failed to promote any response to CLC/CLF in these two cell lines. In contrast, CLC/CLF was
fully active on a BA/F3 cell line expressing the tripartite CNTF
receptor complex on its surface (Fig. 10C). These results indicate that CLC/CLF cannot be substituted by CNTF. In spite of an
in vitro recognition between CLC/CLF and sCNTFR (45), the composite cytokine plus sCNTFR was not able to elicit a functional proliferative response in cell lines expressing only gp130 and LIFR on
their surfaces.
Additional experiments were performed based on the ability of the KB
epidermoid carcinoma cell line to become activated when grown in the
presence of LIF (45). MAP kinase phosphorylation levels were analyzed
in the KB cells (Fig. 11). As
previously shown, KB cells become sensitive to CNTF or CLC/CLF only
when transfected with membrane-bound CNTFR (45). Importantly,
association of sCNTFR to CNTF also induced MAP kinase pathway
activation. In contrast, no activation could be detected when sCNTFR
was used in combination with CLC/CLF. These results clearly established an absolute requirement of the membrane-bound form of CNTFR to generate
a functional response to CLC/CLF composite cytokine.
We reported previously that CNTF and the CLC/CLF heterocomplex act
through the same receptor complex to generate overlapping biological
functions (45). In the present study we have analyzed the cell
signaling mechanisms observed in response to the CLC/CLF composite
cytokine and compared the recruited pathways with those activated by
both CNTF and LIF. Our analysis demonstrates a large overlap between
the pathways activated by LIF, CNTF, and CLC/CLF.
As reported previously for CNTF, the CLC/CLF composite cytokine
preferentially binds to the CNTFR subunit (22, 25, 54). In contrast,
LIFR, known as a binding component for both LIF and cardiotrophin-1,
failed to recognize the dimeric cytokine (49). Similarly, gp130, which
can bind oncostatin M, was not able to contact CLC/CLF directly (19).
The contribution of both gp130 and LIFR to CNTF binding is essential to
increase the affinity of the ligand to the membrane (25, 54). A similar
process might occur for CLC/CLF, because a co-expression of gp130 and LIFR with CNTFR leads to a 3-4-fold increase of the mean fluorescence value recorded by FACS analysis. The use of radiolabeled ligand should
help to determine the affinity constants of CLC/CLF toward the three
components of the functional receptor. By using gp130 neutralizing
antibodies and analyzing the tyrosine phosphorylation content of gp130
and LIFR in response to CLC/CLF, we have shown that the formation of a
gp130-LIFR heterocomplex is essential for the composite cytokine
signaling (45).
In neuroblastoma cells expressing the tripartite CNTF receptor
components on their surface, the CLC/CLF composite cytokine triggered
gp130-LIFR association and tyrosine phosphorylation induced by JAK
tyrosine kinases. As reported previously for related ligands, an
activation of JAK1, JAK2, and TYK2 was observed in response to CLC/CLF
(26-30). According to the respective phenotypes demonstrated in JAK
kinase-deficient mice (56-58), we can hypothesize a prevalent
involvement of JAK1 in gp130-LIFR signaling in response to CNTF and
CLC/CLF (55). Additionally, in Jak1 Following CLC/CLF cell activation and JAK activation, information is
relayed to the nucleus by a number of signaling molecules, including
the STAT3 transcriptional activator, which is quickly recruited by the
second CNTFR ligand. STAT3 is essential for the early development of
the mouse embryo (59). The embryos developed until embryonic day 6 before a rapid degeneration with no obvious mesoderm formation.
Involvement of STAT3 in CNTF signaling was demonstrated initially using
neuroblastoma cell lines or receptor reconstituted systems expressed in
fibroblast cell lines (27, 31). Their functional responses to CNTF have
since been detected in cells of glial origin (60, 61). The present work
led to similar conclusions by demonstrating that STAT3 is also a major signaling protein for the second CNTF receptor ligand. The effect of
the CLC/CLF composite cytokine on glial cells remains to be established. We also detect to a lesser level a STAT1 activation in
CLC/CLF signal transduction. STAT1 gene inactivation in the mouse has
underlined its essential involvement in mediating the antiviral
properties of interferons, although no evidence was found for an
alteration of the responses mediated through the gp130 pathway in these
mice (62).
The tyrosine phosphatase SHP-2 is widely expressed and becomes
tyrosine-phosphorylated after cell stimulation with cytokines (27).
There is some controversy about the role of SHP-2 in tyrosine kinase
signaling and the relative contributions of its SH2 domains, catalytic
domain, and carboxyl-terminal tail to its downstream effects (63).
Thus, SHP-2 may be a positive effector of signal transduction by acting
as an adaptator protein to associate with GRAB2, leading to activation
of the MAP kinase cascade (53, 54). The recruitment of SHP-2 by the
gp130 pathway leads to the transmission of proliferative signals in
various hematopoietic cell systems (64). The use of either a modified
gp130 receptor lacking the SHP-2 binding site or of SHP-2 dominant
negative mutants has demonstrated a negative regulatory role for SHP-2
(65, 66). The dual function of SHP-2 was further reinforced by studies
involving Shp-2 defective mouse embryos, whereby SHP-2 can function as
either a positive or a negative regulator for the MAP kinase
activation, depending on the specific receptor pathway stimulated (63). In the present work, the CLC/CLF composite cytokine strongly recruited SHP-2. Co-precipitation experiments show an association between gp130
and the tyrosine phosphatase most probably through tyrosine 759 of
gp130 as reported before (27). Interestingly, tyrosine 759 has also
been shown to bind the suppressor of cytokine signaling-3 (67, 68). A
number of studies reporting a negative regulatory function of SHP-2
were based on observed increases in gp130-dependent signaling when Tyr579 is mutated (65, 66). Part of the
observation might therefore be linked to the negative regulatory
function of the suppressor of cytokine signaling-3. A recent study
reports that LIFR can directly bind to SHP-2 (69). Similarly, an
association between SHP-2 and LIFR was demonstrated following
activation of the tripartite receptor complex by CLC/CLF. It remains to
be determined whether SHP-2 transduces a positive or a negative
regulatory signal after its recruitment by the CLC/CLF composite
cytokine. A robust recruitment of the PI 3-kinase/AKT pathway was also
observed in response to CNTF and to the second CNTFR ligand. A contact
between p85 PI 3-kinase and SHP-2 was also seen as reported previously
for the IL-6 receptor or the thrombopoietin receptor (39, 70).
AKT recruitment was time-dependent and remains elevated
after a 1-h contact period with the composite cytokine. PI 3-kinase has
previously been reported to act upstream of MAP kinase activation when
the gp130/LIFR complex bound LIF (39). In the present study we observed a rapid induction of ERK1 and ERK2 tyrosine phosphorylation following cellular activation by CLC/CLF. This activation process was transient and entirely disappeared after 20 min of contact, pointing toward the
existence of a negative regulatory loop. This result corroborates the
observation that the AKT pathway was able to down-modulate the Raf
pathway (71). Cross-talk between the MAP kinase and the PI 3-kinase
pathways may switch the biological response between proliferation and
differentiation processes.
In a collaborative study we have previously shown that
sCNTFR was able to promote the CNTF response in cell lines that
only expressed LIFR and gp130 on their surface (23). By performing co-immunoprecipitation experiments we reported the possibility that the
CLC/CLF composite cytokine associates with sCNTFR (45). In the present
work we clearly established that association of CLC/CLF to sCNTFR is
not sufficient to elicit responses in gp130+
LIFR+ cell lines and that an absolute requirement of a
membrane form of CNTFR was necessary to induce a functional response to
the CLC/CLF composite cytokine. This represents a major difference between the activation processes developed by CNTF and CLC/CLF. This
also suggests that, in response to CLC/CLF, the membrane form of CNTFR
would be able by itself to signal through its
glycosylphosphatidylinositol anchorage. Interestingly, similar
observations have been made for a number of
glycosylphosphatidylinositol-linked proteins (72).
In conclusion, we show in neuroblastoma cell lines, both CNTF and
CLC/CLF composite cytokine are able to recruit the STAT3, PI 3-kinase,
and MAP kinase pathways (Fig. 12). This
study reinforces the functional similarity between CNTF and the
composite cytokine defining the long sought CNTF-2 (40, 45). The major
difference between these two cytokines resides in the fact that, in
contrast to CNTF, CLC/CLF requires an absolute requirement of a
membrane-bound form of CNTFR to elicit a functional response. A second
difference is the ability of CLC/CLF to get secreted outside of the
cells, whereas CNTF is mainly released from cells during trauma (73). In contrast to CNTF, CNTFR is highly expressed in the developing embryo
(74), and we are therefore investigating embryonic expression of both
CLC and CLF. Preliminary observations indicate that both components are
expressed during embryonic life. Finally, it will be important to
positively identify CLC/CLF as CNTF-2 and to exclude the possibility of
yet more ligands for the CNTF receptor by demonstrating that a double
knock out for CNTF and CLC encompasses the phenotype resulting
from CNTFR gene disruption.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase encoding expression vector.
Because the expression of large size signaling cytokine receptors on
the membrane is usually weak, receptor expression experiments were
pursued only when more than 80% of the cells were stained blue 48 h after transfection. For recombinant CLC/CLF synthesis, the cells were
cultured for 72 h in serum-free Yssel medium (48).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CNTF receptor expression is necessary for membrane binding of
CLC/CLF
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Fig. 1.
CNTF receptor expression is necessary for
membrane binding of CLC/CLF. COS-7 cells were transfected with
cDNAs encoding the indicated receptors or mock control. After
48 h, the expression of gp130, LIFR, and CNTFR was monitored by
flow cytometry analysis. The cells were incubated with CLC-c-Myc/CLF,
and the membrane cytokine binding was detected with either an
anti-c-Myc, an anti-CLF (AN-F-C6) mAbs, or an isotype control
immunoglobulin.
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Fig. 2.
SK-N-GP and IMR-32 neuroblastoma cell lines
express the tripartite CNTF receptor and bind the CLC/CLF
heterocomplex. The expression of gp130, LIFR, and CNTFR on SK-N-GP
(A) and IMR-32 (B) was monitored by flow
cytometry analysis. The white histograms correspond to the
isotype controls, and the shaded histograms correspond to
receptor detection. Binding of CLC-c-Myc/CLF was detected by incubating
the cell lines with the composite cytokine, which was then detected
using anti-c-Myc or anti-CLF (AN-F-C6) mAbs.
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Fig. 3.
CLC/CLF induces tyrosine phosphorylation of
JAK1, JAK2, and TYK2. The SK-N-GP and IMR-32 neuroblastoma cell
lines were incubated either with or without 20 ng/ml of LIF, CNTF,
CLC/CLF, or IL-2 for 10 min. After lysis in 1% Nonidet P-40, proteins
were immunoprecipitated as indicated, and their tyrosine
phosphorylation levels were analyzed. IP,
immunoprecipitation; WB, Western blot; P.Y.,
phosphotyrosine.
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Fig. 4.
gp130 and LIFR tyrosine phosphorylation in
IMR-32 cells. Following exposure to phosphate-buffered saline or
20 ng/ml of LIF, CNTF, or CLC/CLF, IMR-32 cells were lysed, the
proteins were immunoprecipitated as indicated, and their tyrosine
phosphorylation levels were analyzed. I.P,
immunoprecipitation; WB, Western blot; P.Y.,
phosphotyrosine.
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[in a new window]
Fig. 5.
CLC/CLF induces tyrosine phosphorylation of
STAT1 and STAT3 transducing proteins. SK-N-GP cells were incubated
either with or without 20 ng/ml of LIF, CNTF, CLC/CLF, and IL-2 for 10 min. After lysis in 1% Nonidet P-40, proteins were immunoprecipitated
with an anti-STAT1 polyclonal antibody, and their tyrosine
phosphorylation level were analyzed. SK-N-GP and IMR-32 were stimulated
as described previously. After lysis in 1% Nonidet P-40, the lysates
were subjected to immunoblot analysis with antibodies specific for
activated forms of STAT3 (STAT3-P). The blots were stripped
and reprobed with an antibody recognizing both activated and
nonactivated STAT3 proteins. SK-N-GP cells were activated with 20 ng/ml
of CLC/CLF for 5-80 min. After lysis in 1% Nonidet P-40, the lysates
were subjected to immunoblot analysis as described above.
IP, immunoprecipitation; WB, Western blot;
P.Y., phosphotyrosine.
CLC/CLF induces tyrosine phosphorylation of STAT1 and STAT3
transducing proteins
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[in a new window]
Fig. 6.
Effect of CLC/CLF stimulation on STAT3
transcriptional activity. SK-N-GP neuroblastoma cells were
transiently transfected with a reporter plasmid gene. 48 h
later, the cells were treated with 20 ng/ml of LIF, CNTF, IL-2, or
CLC/CLF for an additional 18 h. The cellular extracts were
prepared and used to directly measure luciferase activity.
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Fig. 7.
CLC/CLF induces tyrosine phosphorylation of
SHP-2 transducing proteins. The SK-N-GP (A) and IMR-32
(B) neuroblastoma cell lines were incubated either with or
without 20 ng/ml of LIF, CNTF, CLC/CLF, or IL-2 for 10 min. After lysis
in 1% Nonidet P-40, the proteins were immunoprecipitated as indicated,
and their tyrosine phosphorylation levels were analyzed. Association of
SHP-2 with others proteins in the SK-N-GP cells (C) was
analyzed using the indicated antibodies for detection. IP,
immunoprecipitation; WB, Western blot; P.Y.,
phosphotyrosine.
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[in a new window]
Fig. 8.
CLC/CLF stimulated phosphorylation of PI
3-kinase and AKT. SK-N-GP cells were incubated either with or
without 20 ng/ml of LIF, CNTF, CLC/CLF, and IL-2 for 10 min. After
lysis in 1% Nonidet P-40, proteins were immunoprecipitated with an
anti-P85 polyclonal antibody, and their tyrosine phosphorylation levels
were analyzed. SK-N-GP and IMR-32 cells were stimulated as described
above. After lysis in 1% Nonidet P-40, lysates were subjected to
immunoblot analysis with antibodies specific for activated AKT
(AKT-P). SK-N-GP cells were activated with 20 ng/ml of
CLC/CLF or CNTF for 5-80 min. After lysis in 1% Nonidet P-40, the
lysates were subjected to immunoblot analysis with antibodies specific
for activated AKT. IP, immunoprecipitation; WB,
Western blot; P.Y., phosphotyrosine.
View larger version (33K):
[in a new window]
Fig. 9.
CLC/CLF induces the phosphorylation of ERK1
and ERK2. SK-N-GP and IMR-32 cells were incubated either with or
without 20 ng/ml of LIF, CNTF, CLC/CLF, and IL-2 for 10 min. After
lysis in 1% Nonidet P-40, the lysates were subjected to
immunoblot analysis with antibodies specific for activated ERK1 and
ERK2. SK-N-GP cells were activated with 20 ng/ml of CLC/CLF or IL-2 for
5-80 min. After lysis in 1% Nonidet P-40, the lysates were subjected
to immunoblot analysis with antibodies specific for activated ERK1 and
ERK2 (ERK1-P and ERK2-P). WB, Western blot.
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[in a new window]
Fig. 10.
Membrane CNTFR expression is required for
the proliferative response of hematopoietic cell lines to CLC/CLF.
TF1 cells and Ba/F3 cells expressing gp130, LIFR, or the tripartite
CNTF receptor (gp130, LIFR, and CNTFR) were seeded in 96-well plates at
a concentration of 5 or 10 × 103 cells/well. Serial
dilutions of tested cytokines were added in triplicate. After a 72-h
incubation period, 0,5 µCi of [3H]thymidine,
was added and 4 h later the incorporated radioactivity was
determined by scintillation counting.
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Fig. 11.
Membrane CNTFR expression is required to
induce the MAP kinase pathway activation in response to CLC/CLF.
KB epidermoid carcinoma cells or CNTFR transfected KB cells were
stimulated for 10 min in the presence of 20 ng/ml cytokine and 500 ng/ml sCNTFR, as indicated. The cells were lysed, and their ERK1 and
ERK2 tyrosine phosphorylation levels were determined. WB,
Western blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice, neurons are
unable to respond to the ligands of the gp130 receptor family and die
by apoptosis, whereas neuronal development was not affected in mice
lacking JAK2, JAK3, or TYK2 (56-58).
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[in a new window]
Fig. 12.
Schematic presentation of signaling pathways
recruitment by CLC/CLF. After binding of CLC/CLF or CNTF to the
tripartite CNTF receptor (gp130, LIFR, and CNTFR), the JAK/STAT, the PI
3-kinase, and the MAP kinase pathways were recruited.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Amy Lundquist and Tyrone Hall for careful reviewing of the manuscript.
![]() |
FOOTNOTES |
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* This work was supported by a grant from the Association Française contre les Myopathies and the Ligue Nationale contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Association Française contre les Myopathies.
¶ Supported by a grant from the city of Angers.
Supported by a grant from the Département du Maine et Loire.
To whom correspondence should be addressed: INSERM E 9928, CHU d'Angers, 4 rue Larrey, 49033 Angers Cedex, France.
Tel.: 33-2-41-35-47-31; Fax: 33-2-41-73-16-30; E-mail:
hugues.gascan@univ-angers.fr.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M101681200
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ABBREVIATIONS |
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The abbreviations used are: CNTF, ciliary neurotrophic factor; CNTFR, CNTF receptor; sCNTFR, soluble CNTFR; LIF, leukemia inhibitory factor; LIFR, LIF receptor; IL, interleukin; CLC, cardiotrophin-like cytokine; PI, phosphatidylinositol; MAP, mitogen-activated protein; CLF, cytokine-like factor-1; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter.
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