(Received for publication, June 11, 1996, and in revised form, November 26, 1996)
From the Laboratoire de Biologie Cellulaire, 4 rue
Larrey, CHU Angers, 49033 Angers Cedex, France and the
Department of Molecular Biology, Genentech Inc.,
South San Francisco, California 94080
Cardiotrophin-1 (CT-1) is a recently isolated
cytokine belonging to the interleukin-6 cytokine family. In the present
study we show that CT-1 activates its receptor expressed at the surface of a human neural cell line by recruiting gp130 and gp190/leukemia inhibitory factor receptor , as shown by analyzing their tyrosine phosphorylation level. Neutralizing antibody directed against gp130 and
reconstitution experiments performed in the COS-7 cell line demonstrate
that gp130-gp190 heterocomplex formation is essential for CT-1
signaling. Analysis of the subsequent activation events revealed that
CT-1 induces and utilizes Jak1-, Jak2-, and Tyk2-associated tyrosine
kinases, which are in turn relayed by STAT-3 transcription factor.
Cross-linking of iodinated CT-1 to the cell surface led to the
identification of a third
component in addition to gp130 and gp190,
with an apparent molecular mass of 80 kDa. Removal of
N-linked carbohydrates from the protein backbone of the
component resulted in a protein of 45 kDa. Our results provide evidence that the CT-1 receptor is composed of a tripartite complex, a situation
similar to the high affinity receptor for ciliary neurotrophic factor.
Interleukin (IL)-61 belongs to a
growing family of cytokines, which includes leukemia inhibitory factor
(LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and IL-11
(Kishimoto et al., 1995). These closely related cytokines
share many common biological properties, such as activation of
hepatocyte transcription (Baumann et al., 1993
), activation
of neural proliferation and differentiation (Yamamory et
al., 1989), and regulation of hematopoiesis (Leary et
al., 1990
; Musashi et al., 1991
). In addition, LIF, CNTF, and OSM display biological properties in the early stages of
embryonic development and allow the in vitro growth of
embryonic stem cells in an undifferentiated state (Smith et
al., 1988
; Conover et al., 1993
). IL-6 and IL-11 are
also important modulators for the immune response by regulating
immunoglobulin secretion (Kishimoto et al., 1995
; Yin
et al., 1992
). The redundancy of their biological properties
is in part explained by the shared use of the common signaling protein,
gp130, in their multimeric receptors. gp130, initially isolated as an
IL-6 receptor signal transducer (Hibi et al., 1990
),
associates with other receptor components to generate high affinity
type receptors for the ligands. This is the case for the gp190/low
affinity LIF receptor (Gearing et al., 1991
), which
associates with gp130 to generate a functional LIF/OSM receptor (Gearing et al., 1992
). For the CNTF receptor, a third
additional component (CNTF
receptor subunit) (Davis et
al., 1991
) interacts with the gp130-gp190 heterocomplex to
generate a high affinity CNTF receptor (Davis et al.,
1993a
). The IL-6 receptor is composed of a gp130 homodimer associated
with an IL-6 binding chain, gp80 (Yamasaki et al., 1988
).
More recently, a binding subunit for IL-11 was also isolated (Hilton
et al., 1994
).
Dimerization of the transducing subunits initiates intracellular
signaling by activating members of cytokine receptor-associated tyrosine kinases, referred to as Jaks (reviewed by Ihle et
al. (1995)). Both gp130 and gp190/LIF receptor can associate Jak1, Jak2, and Tyk2 (Stahl et al., 1994). The information is next
relayed by a family of transcription factors known as STATs (signal
transducers and activators of transcription), which are activated in
the cytoplasm before translocation to the nucleus (Ihle et
al., 1995). IL-6 type cytokines will preferentially activate
STAT-1 and STAT-3 (Stahl et al., 1995
; Lütticken
et al., 1994
).
Recently, a new cytokine named cardiotrophin-1 (CT-1) was isolated
based on its ability to induce cardiac myocyte hypertrophy (Pennica
et al., 1995a; Pennica et al., 1996a
). Analysis
of its amino acid sequence revealed some homologies with LIF and CNTF, suggesting that it belongs to the IL-6 cytokine family. Since then,
additional bioactivities have been ascribed to CT-1, showing that
similar to LIF, CT-1 induces the terminal differentiation of the M1
myeloid cell line, the phenotypic switch in rat sympathetic neurons,
and inhibits the spontaneous differentiation of embryonic stem cells
(Pennica et al., 1995b
). Direct binding of CT-1 to gp190/LIF
receptor and the implication of gp130 as a converter protein in the
mouse CT-1 receptor were also reported (Pennica et al.,
1995b
).
In the present study, we have characterized the receptor subunits
involved in the signaling of the human CT-1 receptor. We found that
gp130 and gp190/LIF receptor associate with a third not yet identified
component of 80 kDa, referred to as the CT-1 receptor subunit
(CT-1R
), to create a triparite receptor for the cytokine. Activation
of the tripartite CT-1 receptor led to the recruitment of Jak1, Jak2,
and Tyk2, which in turn are relayed by STAT-3 to transduce the
information inside the cell.
The SK-N-MC neuroblastoma, KB epidermoid
carcinoma, and COS-7 cell lines from ATCC (Rockville, MD) were
routinely grown in RPMI culture medium supplemented with 10% fetal
calf serum. For the growth of the multifactor-dependent
hematopoietic TF1 and DA1.a cell lines (Kitamura et al.,
1989; Gascan et al., 1990
), the culture media were
supplemented with 1 ng/ml granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor, respectively. The L-CNTF Rec. cell line was derived by stable transfection of the L929 cell line (ATCC) with a cDNA encoding the
human CNTF receptor
subcloned in a pcDNA Neo expression vector
(Invitrogen, San Diego, Ca). Purified human recombinant LIF
(108 units/mg) produced in a Chinese hamster ovary cell
line, E. coli recombinant granulocyte-macrophage
colony-stimulating factor (108 units/mg), and IL-11
(2.5 × 106 units/mg) were kindly donated by Dr. K. Turner (Genetics Institute, Boston, MA). IL-6 (107
units/mg) and OSM (2 × 106 units/mg) were purchased
from Peprotech (Canton, MA). Mouse CT-1 was produced as described
previously (Pennica et al., 1995a
). Rat CNTF was produced as
a GST fusion protein by using pGEX-4T2 gene fusion vector from
Pharmacia (Uppsala, Sweden), before being cleaved with thrombin. B-T6,
B-P4, B-T2, and B-R3 anti-gp130 mAbs were described in detail elsewhere
(Chevalier et al., 1996
). Polyclonal antibodies raised
against gp130 or gp190 were bought from R & D System (Oxon, United
Kingdom). Rabbit anti-Tyk2 and antibodies recognizing the
carboxyl-terminal sequence of gp190 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 UBI (Lake Placid, NY). mAbs recognizing STAT-1 and STAT-3 were
obtained from Transduction Laboratories (Lexington, KY).
Cytokines were added to 104 TF1 or
DA1.a myeloid cells in triplicate in the assay. Cells were incubated
for 72 h before being pulsed with 0.5 µCi of
[3H]thymidine for the last 4 h of the culture
(Fourcin et al., 1994). The KB carcinoma cell line was
plated in triplicate in 96-well plates at a concentration of 50 × 103 cells/well in 150 µl of culture medium containing
serial dilutions of tested cytokines. After 48 h, the supernatants
were harvested, and their IL-6 content was determined by enzyme-linked
immunosorbent assay (Thoma et al., 1994
).
COS-7 cells were
transfected by using the DEAE-dextran method as described previously
(Robledo et al., 1996). After a 48-h culture period,
expression and function of the transfected proteins was studied. The
cDNA encoding the human gp190/LIF receptor
was in pCMX and was
a kind gift of Dr. George D. Yancopoulos (Davis et al.,
1991
). The cDNA encoding the human gp130 transducer was cloned by
polymerase chain reaction from the U266 cell line according to the
published sequences and subcloned in the pCD vector (Robledo et
al., 1996
).
After stimulation, cells
were 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% Triton X-100, Nonidet P-40, or Brij 96, depending on
the experiments (Fourcin et al., 1994). After pelleting
insoluble material and after protein standardization, the supernantants were immunoprecipitated overnight. The complexes were then isolated with beads coupled to protein A, submitted to SDS-PAGE, and transferred onto an Immobilon membrane (Millipore, Bedord, 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, Les Ulis, France) according to the
manufacturer's instructions. In some experiments the membranes were
stripped in 0.1 M HCl-glycine, pH 2.5, for 1 h and
neutralized in 1 M Tris-HCl, pH 7.6, before reblotting.
CT-1,
LIF, and CNTF were iodinated by the two-phase method as described
previously (Gascan et al., 1990). The specific activity of
radiolabeled products was 2,000-9,000 cpm/fmol for CT-1,
10,000-20,000 cpm/fmol for LIF, and 6,000-10,000 cpm/fmol for CNTF.
Cells (0.5-5 × 106) were incubated with the
indicated concentrations of radiolabeled ligand, and the nonspecific
binding component was measured by including a 100-200-fold excess of
unlabeled cytokine. After a 2-h incubation at 4 °C, cell-bound
radioactivity was separated from the unbound fraction. Determination of
affinity binding constants was performed according to Scatchard. For
cross-linking experiments, 2-3 × 106 COS-7 cells,
20 × 106 SK-N-MC cells, or 200 × 106 DA1.a cells were incubated in the presence of 0.5-2
nM 125I-labeled CT-1 with or without a 100-fold
molar excess of unlabeled cytokine. After a 2-h incubation with
agitation at 4 °C, cells were washed extensively and incubated for
an additional 45 min at 20 °C in phosphate-buffered saline, 1 mM MgCl2, 1 mM BS3 (Pierce), 1 mM EGS (Pierce), pH 8.3. After washes, cells were lysed in
lysis buffer containing 1% Brij 96 as described above. Insoluble
material was pelleted, and the supernatants were analyzed by SDS-PAGE
and autoradiography. In some experiments cross-linked products were immunoprecipitated overnight with anti-gp130 or anti-gp190 antibodies before electrophoresis. For the N-linked polysaccharide
analysis, cross-linked material or CT-1 was incubated overnight at
37 °C in the presence of 1 unit of endoglycosidase F (Boehringer,
Mannheim, Germany) per mg of protein in 10 mM phosphate
buffer, 0.5% SDS, 25 mM EDTA, 0.5% 2
-mercaptoethanol,
0.5% Nonidet P-40, pH 6, before being submitted to SDS-PAGE and
autoradiography.
In the present study we have analyzed the
tyrosine phosphorylation events observed in response to CT-1. The
SK-N-MC neuroblastoma cell line was used for these experiments, since
tyrosine phosphorylation was readily detectable in these cells.
Treatment of SK-N-MC cells with CT-1 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.
1A). A similar pattern was observed after
treating the cells with OSM (or LIF), suggesting that the associated
protein was gp190/LIF receptor. This was confirmed by reblotting the
filter with an antibody directed against gp190. A blocking antibody to
gp130 was sufficient to prevent the induction of tyrosine
phosphorylation in both gp130 and gp190 subunit receptors, further
indicating that gp130-gp190 heterodimer formation was required to
induce the phosphorylation of both receptor components (Fig.
1B). In addition, a slight decrease in gp130 electrophoresis mobility was observed upon CT-1 activation, which was probably a result
of the transducer activation as reported previously for IL-3
receptor (Liu et al., 1994
). To determine whether gp130 and
gp190 were sufficient to generate a functional receptor for CT-1,
reconstitution experiments using cDNAs encoding these subunits were
carried out in the COS-7 cell line (Fig. 1C). Individual expression of gp130 or gp190 did not allow the induction of tyrosine phosphorylation of these components by CT-1. In contrast, receptor activation was readily detectable when both subunits were co-expressed in the COS-7 cells, indicating that gp130-gp190 heterocomplex formation
was essential for CT-1 signaling.
CT-1 Signaling Pathway Involves Jak1, Jak2, Tyk2 Tyrosine Kinases, and STAT-3 Transcription Factor
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 (Stahl et
al., 1994). This led us to investigate whether the Jak-Tyk kinases
are involved in signaling initiated by CT-1. Fig. 2,
A-C show that tyrosine phosphorylation of Jak1-, Jak2-, and
Tyk2-signaling proteins is induced after treatment of SK-N-MC cells by
CT-1. Tyk2 phosphorylation observed in response to CT-1 is weaker than
the signals detected for Jak1 and Jak2 as previously reported for the
other members of the IL-6 family. Nevertheless, immunoblots of lysates
revealed a clear expression of Tyk2. Thus, these data indicate that the
signaling cascade induced by CT-1 is similar to that induced by the
related members of the family (Guschin et al., 1995
).
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. As
shown in Fig. 2, D-E, stimulation of the SK-N-MC cells with
CT-1 elicits activation of STAT-3 protein. We failed to detect an
induction of tyrosine phosphorylation in STAT-1 after CT-1 activation
(Fig. 2D), sustaining the notion of a specific recruitment
of STAT-3 by CT-1.
Moreover, immunoprecipitation of the gp130 receptor allowed the
co-precipitation of the LIF receptor but also of some additional tyrosine-phosphorylated proteins with molecular masses of 110-130 kDa
and in the range of 80-90 kDa (Fig. 3A).
Immunostaining of the blots with antibodies recognizing the Jak family
members demonstrated that the Jak1 and Jak2 kinases could be
co-precipitated with gp130, and were preassociated to gp130 in the
absence of ligand. Tyk2 did not directly contact gp130 even after
induction of gp130-gp190 heterodimerization (Fig. 3, D-F).
Interestingly, the STAT-1 signaling protein was preassociated with
gp130, but no variation in its detection or activation level could be
observed after CT-1 treatment of the cells. In contrast, CT-1
stimulation induced a strong association of STAT-3 to the gp130-gp190
heterocomplex, but the detected, associated form of STAT-3 was weakly
tyrosine-phosphorylated (Fig. 3, A and C), which
is in striking contrast with the status of STAT-3 directly
immunoprecipitated from the cytoplasmic extract (Fig. 2E).
This indicates that STAT-3 was immediately released from the transducer
receptors after activation.
CT-1 Cell Surface Binding
Scatchard analysis of saturation
binding data was carried out in the SK-N-MC cell line, using
concentrations of CT-1 ranging from 15 pM to 5 nM. Binding of 125I-CT-1 was specific and
saturable and displayed high and low affinity sites in the SK-N-MC cell
line (Fig. 4A). A biphasic Scatchard plot was
observed with Kd values of 400-500 pM
and 4 nM, indicating approximately 1,100-1,500 and 6,000 sites per cell, respectively. To assess the binding capacity of the
gp130 and gp190 components, reconstitution experiments were performed
in the COS-7 cell line. gp190-transfected cells bound both radiolabeled CT-1 and LIF with an affinity constant of about 109
M (Table I). In contrast, the expression of
gp130 alone led to a receptor that failed to bind CT-1 in an efficient
way, indicating that the contribution of gp130 to CT-1 binding was not
essential. Co-expression of gp130 and gp190 receptor subunits in COS-7
cells gave rise to high affinity receptors for both CT-1 and LIF
(Kd = 200-470 pM) (Table I and Fig.
4B), in agreement with the recruitment of the two receptor
subunits to generate a functional response as observed in Fig.
1C. We further analyzed the potential cross-competitions between CT-1 and the other members of the IL-6 family of cytokines toward their receptors. The experiments were performed on the SK-N-MC
cell line, which expresses functional high affinity receptors for all
described cytokines belonging to this group, as shown by Scatchard
analysis and tyrosine phosphorylation assays of gp130 (Fig.
5A) (Chevalier et al., 1996
).
Cells were incubated in the presence of 1 nM radiolabeled
CT-1 and a 200-fold excess of the putative competitors. Fig.
5B shows that bound radioactivity was displaced by an
exogeneous addition of cold CT-1 and also by LIF and OSM. Cytokines
using a homodimer of gp130 in their transducing machinery, such as
IL-6, IL-11, or granulocyte-macrophage colony-stimulating factor, known
to use a different receptor complex, did not interfere with CT-1
binding. As to the effect of CNTF on the binding of CT-1 to its
receptor, only a slight displacement of the radiolabeled protein was
observed. Reciprocal experiments conducted with 125I-CNTF
confirmed that CNTF and CT-1 did not cross-compete or cross-competed only in a very marginal way, whereas competition between LIF and CNTF
was observed as reported previously (Robledo et al., 1996
) (Fig. 5C).
|
Identification of a Third
We next employed cross-linking of iodinated CT-1
to the SK-N-MC cell surface to determine whether the cytokine directly
contacted gp130 and gp190. Three labeled bands were detected
corresponding to the molecular masses of 190-210 kDa, 130-150 kDa,
and 80 kDa (Fig. 6A). The identity of two
higher molecular mass bands was confirmed by immunoprecipitating the
cross-linked products with antibodies raised against either gp190 or
gp130. Similar patterns were obtained, demonstrating that gp190 and
gp130 were components of the cross-linked products (Fig.
6B). In addition, gp130, gp190, and gp80 remained
associated, showing that these proteins tightly interacted to generate
a tripartite complex. The observed bands were competed by an excess of
unlabeled CT-1 or LIF, which is consistent with the results obtained in
the radioreceptor experiments (Fig. 5A). Similar experiments
using radiolabeled LIF were also carried out, and using these
experimental procedures a single band that corresponded to gp190
(190-210 kDa) and was competed by an excess of LIF or CT-1 was
observed (data not shown).
To rule out the possibility of the 80-kDa band representing a degradation product of the gp130 or gp190 components, the experiments were performed in the presence of protease inhibitors. In additional experiments, incubation times with the cross-linking agents were increased up to 2 h, and an aliquot of the samples was incubated at 37 °C for 24 h before being electrophoresed and compared with the conventional preparations. No variation in the ratio of the three cross-linked components could be observed, showing that gp80 did not result from a proteolytic fragmentation of gp130 or gp190 (data not shown). In addition, gp130 and gp190 were activated in the presence of CT-1 separated on SDS-PAGE and blotted as described in Fig. 1A. Staining of the membranes with polyclonal antibodies recognizing either gp130 or gp190 did not show a signal in the range of 80 kDa, indicating that the identified protein was not a clipped or a soluble product of the transducing receptor components (data not shown).
The 80-kDa subunit of the CT-1 receptor was further characterized by
analyzing its carbohydrate content after removing the N-linked sugars from the protein backbone. A preliminary
experiment indicated that N-linked polysaccharides account
for 2-3 kDa of the CT-1 molecular mass (Fig. 6C,
right part), which is in agreement with the presence of a
single potential N-glycosylation site in the CT-1 sequence
(Pennica et al., 1995a). Treatment of the
125I-CT-1 cross-linked receptor subunits with
endoglycosidase F led, as expected, to a faster electrophoretic
mobility of the products. Nevertheless, only a slight shift in
gp190/LIF receptor molecular weight was detected, although 20 potential
N-glycosylation sites are present in its amino acid sequence
(Gearing et al., 1991
). This result suggests that either the
enzyme digestion was incomplete or that electrophoresis resolution was
not sufficient to fully discern a molecular weight shift for large
proteins. After treatment of gp130 with endoglycosidase F, a molecular
mass of 100-110 kDa was observed as previously reported (Taga et
al., 1989
). Interestingly, the polysaccharide moiety of the CT-1
receptor
subunit represented about 35 kDa, leading to an
N-deglycosylated protein with an apparent molecular mass of
45 kDa. The COS-7 cell line expresses functional high affinity binding
sites for CT-1, when cotransfected with cDNAs encoding gp130 and
gp190 (Fig. 4B and Table I). In order to determine whether
the 80-kDa component was present in the COS-7 cell line, cross-linking
experiments were conducted in the COS-7 cells transfected with the
appropriate cDNAs. In the nontransfected cells or in COS-7 cells
transfected with a cDNA encoding gp130, a slight cross-linking of
CT-1 to endogeneous LIFR
could be observed in some instances (Fig.
7A). Introduction of gp190 allowed a clear detection of CT-1 associated to LIFR
. In COS-7 cells co-transfected with cDNAs encoding gp130 and gp190, the three components could be
cross-linked, indicating that the 80-kDa subunit is endogenously present but that expression of both transducing receptor components was
required to detect a CT-1R
cross-linking. After immunoprecipitation with antibodies directed against gp130 or gp190, the cross-linked products remained associated, indicating that the three receptor components tightly interacted in the presence of the ligand (Fig. 7B). These results could explain the presence of high
affinity binding sites for CT-1 observed in the COS-7 cells transfected with both cDNAs (Table I).
CT-1R
To evaluate any
relationship between CT-1R and the previously described
components for IL-6, IL-11, and CNTF receptors, a series of experiments
were carried out (Fig. 8). CT-1 failed to bind CNTF
receptor expressed in a fibroblast cell line, whereas 125I-CNTF bound to its
receptor component expressed in
the same cell line (Fig. 8, A and B). The
IL-6-sensitive hematopoietic TF1 cell line was also found to
proliferate in response to CT-1 and expressed its tripartite receptor.
B-R6 mAb recognized the gp80/IL-6 receptor and inhibited its
association to gp130 but not IL-6 binding (Fourcin et al.,
1994
). CT-1R
was different from the gp80/IL-6 receptor, since B-R6
mAb only abrogated the response of the TF1 cell line to IL-6 but not to
CT-1 (Fig. 8C). Since the CT-1 tripartite receptor is
present in the KB epidermoid cell line (data not shown), we compared
the induction of IL-6 secretion by culturing this line in the presence
of CT-1 or IL-11. CT-1 induced IL-6 secretion, but IL-11 remained
without effect, indicating that the IL-11 receptor
subunit was not
an essential component for the CT-1 receptor (Fig. 8D).
Collectively, these results indicate that CT-1R
is different from
the
components involved in the formation of the IL-6, IL-11, or
CNTF receptors.
CT-1R
Scatchard analysis of CT-1 binding to different cell
types led to the identification of the murine hematopoietic Da1.a cell line (Gascan et al. 1990), which bound CT-1 only with a low
affinity binding component (Kd = 5-7
nM) (Fig. 9A). In contrast, the
DA1.a cell line displayed high affinity binding sites for both human
and mouse LIF (Kd = 40-50 pM) and
strongly proliferated in response to LIF, indicating the cell surface
expression of the gp130-LIFR
heterocomplex (Gascan et al.
1990
) (Fig. 9C and data not shown).
Cross-linking of iodinated murine CT-1 to the surface of the DA1.a
cells allowed the detection of a single product with an apparent
molecular mass of 170 kDa, corresponding to the murine form of LIFR
(Fig. 9B). Compared with the CT-1 cross-linking experiments
carried out with the SK-N-MC cell line, a 10-fold higher DA1.a cell
number was used, and the exposure times were lengthened from 4 to 21 days, further indicating that CT-1R
was probably not expressed in
the DA1.a cell line.
Analysis of tyrosine phosphorylation events in the DA1.a cells in
response to CT-1, LIF, or OSM did not allow the detection of a gp130 or
LIFR activation. We recently reported a similar situation for the
TF1 cell line, which proliferated in the presence of the IL-6 type
cytokines, but where the use of 109 cells only allowed the
detection of a liminar activation of the gp190-gp130 complex (Chevalier
et al., 1996
). These results are agreement with the
observations showing that tyrosine phosphorylation of a
65 gp130
truncated mutant was not required for proliferation (Murakami et
al., 1991
). To further analyze the functional response of the
DA1.a cell line we have studied the proliferation of the cells in
response to CT-1 and LIF. Fig. 9C shows that in the KB cells, expressing the tripartite CT-1 receptor complex, a 4-fold decrease in the sensitivity of the response to the cytokines was observed (LIF EC50 = 80 pg/ml; CT-1 EC50 = 330 pg/ml). In contrast, the DA1.a cell line was 200-fold less sensitive to
CT-1 compared with LIF (LIF EC50 = 5 pg/ml; CT-1
EC50 = 1,000 pg/ml).
Together, these results indicated an apparent lack of CT-1R
expression in the DA1.a cell line, resulting in a low affinity binding
of the cytokine to the cell surface and a proliferative response
observed only in the presence of high concentrations of CT-1. This
situation is very similar to the one we observed previously for the
hematopoietic TF1 cell line in response to CNTF (Davis et
al., 1993b
).
Cardiotrophin-1 was named and isolated based on its ability to
induce cardiac myocyte hypertrophy in vitro (Pennica
et al., 1995a; Pennica et al., 1996a
). Analysis
of its amino acid sequence showed some similarities with the members of
the IL-6 family of cytokines, in particular with LIF and CNTF. The
availability of purified recombinant CT-1 allowed the characterization
of the mouse CT-1 receptor. Involvement of the common
signal-transducing component, gp130, and LIF receptor in the CT-1
receptor was demonstrated (Pennica et al., 1995b
). In the
present study we have characterized the human CT-1 receptor and its
signaling machinery. Furthermore, reconstitution experiments and
cross-linking of the radiolabeled cytokine to its receptor revealed
that the CT-1 receptor is a tripartite complex, a situation comparable
with that of the high affinity CNTF receptor.
Tyrosine phosphorylation analysis of CT-1 receptor components and
reconstitution experiments carried out in the COS-7 cell line led to
the conclusion that gp130 associates with the gp190/LIF receptor to
create a functional receptor able to transduce CT-1 signaling inside
the cell. Transfection experiments revealed that human gp190 is a major
binding component for CT-1 as observed for the mouse receptor (Pennica
et al., 1995b), whereas gp130 behaves more like a convertor
protein, a situation reminiscent of the high affinity LIF receptor,
where LIF preferentially contacts gp190 before recruiting the
gp130-transducing chain (Gearing et al., 1992
). By using a
neutralizing antibody directed against gp130 and performing COS-7
transfection experiments, we show that formation of a gp130-gp190
heterocomplex is essential for CT-1 signaling.
Analysis of the subsequent activation events indicates that CT-1
binding to gp130-gp190 transducing complex leads to the activation of
Jak1, Jak2, and Tyk2 kinases, as reported for the other members of the
IL-6 family (Stahl et al., 1994; Lütticken et
al., 1994
; Guschin et al., 1995
). The information is
next relayed to the nucleus by the STAT-3 transcriptional activator,
whose association to the transducing complex is strongly enhanced by
the binding of CT-1, before being released after tyrosine
phosphorylation in the first 10 min following contact with the
cytokine. We failed to detect an implication of STAT-1 in CT-1
signaling, which might represent a difference with the IL-6 pathway or
could also be the result of tissue variations. Several recent studies
have reinforced the notion that STAT-3 is a major transcriptional
factor phosphorylated in response to gp130-gp190 complex activation and
that STAT-1 recruitment seems more restricted to IL-6 stimulation
(Boulton et al., 1995
; Zhang et al., 1995
;
Guschin et al., 1995
). Very recently, STAT-1 gene
inactivation in the mouse has underlined its essential implication in
mediating the antiviral properties of interferons (Meraz et
al., 1996
; Durbin et al., 1996
). No evidence for an
alteration of the physiological responses dependent upon the IL-6
cytokine family was detected in the STAT-1-deficient mice.
CT-1 binding experiments defined high affinity and low affinity binding
sites. Since gp190 behaves as a major binding component for CT-1, we
can hypothesize that relative expression levels of each receptor
component might affect the apparent affinity of CT-1 for the cell
surface, as previously observed for LIF or CNTF receptors (Robledo
et al., 1996). Cross-linking of CT-1 to the SK-N-MC cell
line led to the identification of a third entity with an apparent
molecular mass of 80 kDa. Reconstitution experiments carried out in the
COS-7 cells suggest that the 80-kDa product/CT1-R
interacted very
weakly, or even did not directly interact with CT-1 when expressed
alone. In contrast, in the presence of the cross-linked ligand, the
tripartite CT-1 receptor components can be co-precipitated with an
antibody raised against either gp130 or gp190. This result indicates
that the CT-1 receptor subunits, when activated, can tightly interact
together, as reported also in the case of the tripartite CNTF receptor
(Stahl et al., 1993
).
Interestingly, cross-linked CT-1 could be displaced from gp80 subunit
by adding excess unlabeled LIF, suggesting that gp80 might be involved
in the formation of the LIF receptor as well. Experiments by using the
DA1.a LIF-sensitive cell line indicate that this latest cell line bound
CT-1 with a low affinity (Kd > 5 nM)
and bound LIF with a high affinity (Kd = 40-50 pM). We failed to observe an expression of CT-1R on the
DA1.a cell surface. Cross-linking experiments only allow the detection of a CT-1 binding to LIFR
, a situation similar to that reported previously for the M1 myeloid cell line (Pennica et al.,
1995b
). In addition, the DA1.a cell line appears to be 200-fold less
sensitive to CT-1 compared with LIF. This suggests that the gp80
subunit might not be required for the formation of high affinity LIF
receptor and that the identified gp80 component could represent an
chain conferring an increased sensitivity and specificity to CT-1. A parallel can be made with the TF1 cell line, which did not express CNTF
receptor
and only responded to the addition of high CNTF concentrations to the cultures (Davis et al., 1993a
).
Interestingly, the addition of soluble CNTF receptor
to the
cultures could increase the sensitivity of the cells to CNTF to reach a
level identical to the one observed in the presence of LIF. We can
hypothesize that a similar complementation mechanism might occur for
the DA1.a cells and CT-1 if a soluble form of CT-1R
could be
found.
Members of the IL-6 cytokine family have been shown to modulate the
phenotype and survival of neuronal cells. The ability of CT-1 to induce
chick ciliary neuron survival was, however, 1000-fold less potent than
the response observed to CNTF (Pennica et al., 1995b). This
result might be explained by a lack of CT-1R
expression in these
neurons. On the other hand, CNTF receptor
gene inactivation
experiments led to a profound anomaly in motoneuron development,
whereas CNTF-deficient mice displayed only mild effects on behavior and
physiology (DeChiara et al., 1995
; Masu et al., 1993
). These studies support the idea that an alternate ligand for the
CNTF receptor
exists. We have investigated the possibility of CT-1
being a new ligand for CNTF receptor
. The fact that cells
transfected with a cDNA encoding this subunit failed to bind CT-1
and that soluble CNTF receptor
did not potentiate the CT-1
response2 support the notion that CT-1 is
not an alternate ligand for the CNTF receptor
component. Two other
-specific binding components, interacting with IL-6 and IL-11, have
also been identified. The use of an antibody decoupling IL-6
receptor/gp130 interaction and the absence of response of the
CT-1-sensitive KB cell line to IL-11 indicate that CT-1R
is
different from the previously identified
subunits.
A very recent paper from Pennica et al. (1996b) demonstrated
that the survival of motoneurons to CT-1 involves, in addition to gp130
and LIFR
, a third additional component sensitive to a phospholipase
C treatment. This result strongly suggests that CT-1R
links to the
membrane through the GPI motif.
The observed situation for the tripartite CT-1 receptor is similar to
the one described for the CNTF high affinity receptor (Davies et
al., 1993). In this case, CNTF and its associated component
contacts gp130. These initial interactions are followed by gp190
recruitment for signaling. For the CT-1 receptor, a mirror image is
observed, where CT-1 probably contacts gp190 and its
component
first before generating the tripartite form of the receptor by engaging
gp130. This notion should be reinforced by the molecular cloning of the
CT-1 receptor
component.
We thank Todd A. Swanson for CT-1 protein purification and Jean-Paul Gislard for assistance with the figures.