From the Biochemisches Institut, Christian Albrechts
Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany
and the
College of Life Sciences, Zhejiang University,
Hangzhou 310027, Peoples Republic of China
Received for publication, October 1, 2002, and in revised form, December 20, 2002
Human ciliary neurotrophic factor (CNTF) is a
neurotrophic cytokine that exerts a neuroprotective effect in multiple
sclerosis and amyotrophic lateral sclerosis. Clinical application of
human CNTF, however, was prevented by high toxicity at higher dosages. Human CNTF elicits cellular responses by induction of a receptor complex consisting of the CNTF
-receptor (CNTFR), which is not involved in signal transduction, and the
-receptors gp130 and leukemia inhibitory factor receptor (LIFR). Previous studies with rat
CNTF demonstrated that rat CNTF is unable to interact with the human
interleukin-6
-receptor, whereas at high concentrations, it can
directly induce a signaling heterodimer of human gp130 and human LIFR
in the absence of the CNTF receptor. Here, we demonstrate that human
CNTF cannot directly induce a heterodimer of human gp130 and LIFR.
However, human CNTF can use both the membrane-bound and the soluble
human IL-6R as a substitute for its cognate
-receptor and thus widen
the target spectrum of human CNTF. Engineering a CNTFR-specific
human CNTF variant may therefore be a prerequisite to improving the
safety profile of CNTF.
 |
INTRODUCTION |
Ciliary neurotrophic factor
(CNTF)1 was identified as a
survival factor for chick ciliary neurons (1, 2) and belongs to the
interleukin (IL)-6 family of structurally related hemato- and
neuropoietic cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF),
oncostatin M (OSM), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine
(CLC, also designated novel neurotrophin-1/B-cell-stimulating factor-3
(NNT-1/BSF-3)) (3-5). Cellular responses to CNTF and IL-6 type
cytokines are elicited by different multiunit receptor complexes that
always include the membrane-spanning 130-kDa glycoprotein, gp130 (3,
6). CNTF first binds in a 1:1 stoichiometry to the GPI-anchored CNTF
receptor (CNTFR
), which is not involved in signal transduction (7,
8). Binding of CNTF to the membrane-bound or soluble CNTFR
induces a
heterodimer of the signal transducing
-receptors gp130 and LIF
receptor (LIFR), which triggers intracellular signaling cascades.
Similar to CNTF, CT-1 and CLC bind to
-receptors before inducing a
gp130/LIFR heterodimer (9, 10), whereas LIF and OSM directly induce a
gp130/LIFR heterodimer (11). OSM may also signal via a second
heterodimer of gp130 and the OSM receptor (12). In contrast to the
aforementioned cytokines, IL-6 and IL-11 induce a gp130 homodimer after
binding to specific
-receptors, the IL-6R
and the IL-11R
(3).
The bewildering variety of receptor complexes of the IL-6 family,
however, funnels into activation of an only limited number of
intracellular signaling cascades including the JAK/STAT, Ras/MAP
kinase, and the phosphatidylinositol 3-kinase pathway (6, 13).
The expression of CNTF is restricted to Schwann cells in the peripheral
and astrocytes in the central nervous system (CNS) (14, 15). Although
involved in neuronal differentiation processes in vitro
(16), CNTF is primarily considered as a lesion factor because of the
lack of a signal peptide in the CNTF gene and its immunohistochemically determined cytosolic localization (1). Several
studies showed that CNTF prevents degeneration of axotomized peripheral
motor neurons and also retrograde cell death of neurons in thalamic
nuclei after dissection of intracerebral neuronal circuits (17, 18).
Intraperitoneal implantation of a CNTF-producing cell line improved
survival in a mouse model of motor neuropathy (19), and in different
models of Huntington's disease, CNTF exerted a neuroprotective effect
on striatal cells (20-22). CNTF also enhanced the survival of sensory,
hippocampal, and cerebellar neurons (23-25) and increased survival of
retinal photoreceptors in animal models of retinal degeneration
(26-28). On denervated rat skeletal muscles, CNTF reduced
denervation-induced atrophy and increased muscular strength (29,
30).
CNTF knock-out (
/
) mice display a mild phenotype showing
progressive loss of motor neurons and reduced muscular strength only in
the adult, not in childhood (31). Cross-breeding of CNTF
/
mice with LIF
/
mice resulted in a
much more prominent phenotype, suggesting that LIF also exerts a
physiological neurotrophic effect (32). In contrast to
CNTF
/
mice, CNTFR
/
mice died within
24 h postnatally because of a suckling defect caused by severe
motor neuron deficits (33). This indicated the existence of a second
ligand for the CNTFR. Surprisingly, the knock-out of an orphan
receptor, NR6, resulted in a phenotype almost identical to that of
CNTFR
/
mice (34). This enigma was resolved by the
discovery, that CLC is the second ligand to the CNTFR and that NR6 is
identical to another orphan receptor, CLF, which is essential for
transport to the cell surface and secretion of CLC (9, 35, 36).
In contrast to mice, a null mutation of the CNTF gene found
in high prevalence in the Japanese population was not causally related
to neurological diseases (37). However, recent studies strongly suggest
that the absence of CNTF modifies the course of some important
neurological diseases. Postmortem spinal cord samples from patients
with amyotrophic lateral sclerosis (ALS) showed decreased levels of
CNTF (38), and in patients with ALS due to a mutation of the superoxide
dismutase gene, CNTF deficiency was associated with an earlier onset
and a more severe course of the disease (39). CNTF-deficient patients
also displayed a significantly earlier onset of multiple sclerosis (MS)
and more severe symptoms (40). Apparently, CNTF exerts a protective
effect in demyelinating disease by preventing apoptosis of
oligodendrocytes (41). CNTF also exerts an anti-inflammatory effect in
the central nervous system by inhibition of TNF
production and
activation of the hypothalamic pituitary axis (42). The association of CNTF deficiency with clinically more severe MS in humans is all the
more surprising, since a model disease of MS in mice, experimental autoimmune encephalitis, cannot be elicited in IL-6
/
mice (43-46).
In recent mouse studies, CNTF activated hypothalamic satiety centers
independently of leptin and reduced obesity and diabetes associated
with overweight or leptin resistance (47, 48). At concentrations
sufficient for weight reduction, no intolerable side effects occurred.
It was suggested that CNTF could therefore be the basis for new
strategies to combat human obesity provided the chemical structure of
CNTF could be redesigned to reduce or eliminate side effects (49). In a
toxicity trial of CNTF in humans, cachexia, aseptical meningitis,
reactivation of virus infections, and respiratory failure were observed
at high doses (>5 µg/kg/day) after subcutaneous injection of CNTF
(50). In a placebo-controlled trial of CNTF in patients with ALS, the
dosage was therefore limited to 5 µg/kg/day, where, however, no
beneficial clinical effects were observed compared with placebo
(51).
In experiments with human HepG2 hepatoma cells, rat CNTF, which has an
85% amino acid identity to human CNTF, induced expression of acute
phase proteins (52, 53). It was explicitly shown that the rat CNTF
activity on human HepG2 cells was not relayed via the human IL-6R (52),
although apparently, rat CNTF stimulates the acute phase response on
primary hepatocytes after binding to the rat IL-6R (54). At high
concentrations, rat CNTF induces an active heterodimer of human gp130
and human LIFR even in the absence of the CNTFR
(11). We have
recently observed activity of recombinant human CNTF at high
concentrations on HepG2 cells, which do not express the CNTFR (53), but
did not observe a similar effect on murine BaF/3 cells stably
transfected with human gp130 and human LIFR. The discrepancy prompted
the present study, which demonstrates that in contrast to rat CNTF,
human CNTF is unable to elicit a heterodimer of human gp130 and LIFR in
the absence of an
-receptor. However, the human IL-6R can serve as a
substitute
-receptor for human CNTF in the assembly of a functional
receptor complex. Molecular modeling revealed the molecular basis for
the difference between human and rat CNTF in binding to the human IL-6R.
 |
EXPERIMENTAL PROCEDURES |
Cells and Reagents--
Human HepG2 hepatoma were bought from
ATCC (Manassas, VA) and were routinely grown in RPMI 1640 or
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. The different BAF/3 cell lines (BAF/3-[gp130,IL-6R],
BAF/3-[gp130,LIFR], BAF/3-[gp130,LIFR,IL-6R], BAF/3-[gp130,LIFR,CNTFR]) used in this study have been described before (55). The murine monoclonal antibody (mAb) ab6276
(anti-
-actin) was bought from abcam (Cambridge, UK), the polyclonal
rabbit antibody anti-phospho-STAT3 (Tyr-705) was from Cell Signaling
Technology (Beverly, CA), polyclonal horseradish peroxidase-coupled
goat anti-rabbit and anti-mouse antibodies were obtained from Pierce.
The neutralizing anti-IL-6 receptor mAb PM-1 has been described before
(56). Soluble IL-6R and CNTFR were obtained from R&D Systems
(Wiesbaden, Germany). [3H]thymidine was from Amersham
Biosciences. Brij-96 and all other reagents were bought from
Sigma-Aldrich. OSM was cloned into the pET-14b bacterial expression
vector (Novagen, Schwalbach, Germany) and purified with a Ni-chelate
column (Amersham Biosciences). All other human or designer proteins
used in this study were produced in Escherichia coli
bacteria as described before (55, 57, 58).
Bioassays--
Proliferation of the transfected BAF/3-[gp130]
cell lines in response to human IL-6, human CNTF, human LIF, and the
designer cytokine IC7 was measured in 96-well microtiter plates. The
cells were exposed to test samples for 72 h and subsequently
pulse-labeled with [3H]thymidine for 4 h.
Proliferation rates were measured by harvesting the cells on glass
filters and determination of the incorporated radioactivity by
scintillation counting. Each proliferation assay was performed at least
three times in triplicates.
Analysis of STAT3 Phosphorylation--
A lysis buffer consisting
of 50 mM Tris, pH 7.5, 100 mM NaCl, 50 mM sodium fluoride, 3 mM sodium orthovanadate,
1% Brij-96, and proteinase inhibitors (1 mM
phenylmethylsulfonyl fluoride, one tablet of the Roche proteinase
inhibitor mixture) was prepared. Transfected BaF/3-[gp130,LIFR,IL-6R]
cells were starved for 4 h in serum-free Dulbecco's minimal
essential medium supplemented with penicillin and streptomycin. After
stimulation with cytokines (50 ng/ml) for 10 min at 37 °C, cells
were pelleted and resuspended in 1 ml of lysis buffer. An aliquot of 40 µl was subjected to SDS-PAGE and blotted to a polyvinylidene
difluoride membrane (Amersham Biosciences). The membranes were
incubated with the anti-phospho-STAT3 antibody before being labeled
with a secondary antibody coupled to peroxidase. Subsequently, the
membranes were developed using the Amersham Biosciences ECL
chemiluminescence kit. HepG2 cells were grown on 24-well plates (TPP,
Biochrom, Berlin, Germany) starved overnight in serum-free medium
before stimulation for 10 min with the indicated cytokines. The
supernatants were removed, and the cells lysed by addition of Laemmli
buffer (2× concentrated). The lysate was subsequently Western blotted
as above. Loading was controlled by a Western blot against
-actin.
CD Spectra--
CD spectra of all cytokines produced were
recorded with a Jasco J-720 spectropolarimeter (Japan Spectroscopic
Co., Ltd., Tokyo, Japan) to check correct refolding. The instrument was
calibrated according to Chen and Yang (73). The spectral
bandwidth was 2 nm. The measurements were carried out at a temperature
of 23 °C, the solvent was phosphate-buffered saline, pH 7.4, throughout.
Determination of the Affinity Constants of IL-6 and CNTF Binding
to the IL-6R by Plasmon Resonance--
IL-6R-Fc was covalently
immobilized to a carboxymethyl dextran matrix (Fisons, Loughborough,
UK) at 28.0 µg/ml for 5 min in 10 mM sodium acetate
buffer, pH 5.0, as recommended by the manufacturer. Binding experiments
were performed at controlled temperature (25 °C) with different
concentrations of purified IL-6 and CNTF protein using the
IASYSTM optical biosensor (Affinity Sensors, Cambridge,
UK). Association was monitored for at least 2 min, the sample was
replaced by phosphate-buffered saline/0.05% Tween 20, dissociation was
monitored, and the cuvette was equilibrated again in phosphate-buffered
saline/0.05% Tween 20. Association and dissociation affinograms were
analyzed by nonlinear regression with the FAST fit (Fisons) software,
which uses the Marquardt-Levenburg algorithm for iterative data fitting.
Molecular Modeling--
The model of the CNTF/IL-6R complex was
built using the x-ray structure of CNTF (Ref. 59, PDB accession code:
1cnt) and the model of the IL-6/IL-6R complex (60) as a template. In a first step the CNTF molecule was fitted onto the IL-6 model (using only
the C
positions of helices A and D). The next steps were performed
in an iterative manner. First, the interaction area was inspected for
overlapping side chains. Unfavorable contacts were then eliminated by
rotating them properly. Second, the accessible surface was calculated
for this complex to find cavities in the interaction area. If possible
these cavities were filled by adjustment of side chains from their
neighborhood. These complexes were then energy-minimized using the
steepest descent algorithm implemented in the GROMOS force field (61)
and again analyzed for unfavorable contacts and cavities in the
interaction area. This procedure was repeated until a low energy
conformation of the complex was reached. Accessible surfaces were
calculated using the algorithm implemented in the software package
WHATIF (62). For graphical representation the Ribbons program was used
(63). All programs were run on a Silicon Graphics Indy workstation.
 |
RESULTS |
Murine BaF/3-cells do not express IL-6-type cytokine receptors,
but proliferate in response to human IL-6 type cytokines upon transfection of appropriate human receptors. To analyze the interaction of human CNTF with different receptors of the IL-6 family, we made use
of a set of murine BaF/3 cells stably transfected with different
combinations of human IL-6-type receptors (Fig.
1). gp130 and LIFR are expressed at equal
levels in these cell lines (55). Human CNTF-stimulated proliferation of
BaF/3-cells stably transfected with gp130, LIFR, and CNTFR (Fig.
1A), but even at very high concentrations up to 10 µg/ml,
it was inactive on BaF/3-[gp130,LIFR] cells that do not express the
CNTFR (Fig. 1B), suggesting that the CNTFR is an absolute
requirement for activity of human CNTF. Similarly, CNTF was inactive on
BaF/3-[gp130,IL-6R] cells, which lack the LIFR and the CNTFR (Fig.
1C). Unexpectedly, however, CNTF showed activity on
BaF/3-[gp130,LIFR,IL-6R] cells that expressed the human IL-6R instead
of the CNTFR (Fig. 1D). Maximal activity of CNTF was
achieved at concentrations exceeding 250 ng/ml, but a clear effect of
CNTF was already discernible at 10 ng/ml. LIF, IL-6, and the designer
cytokine IC7, which induces a gp130/LIFR heterodimer after binding to
the IL-6R (55), stimulated proliferation with ED50 of 0.6, 0.9, and 0.5 ng/ml, whereas an about 42-fold higher CNTF concentration
(ED50 = 30 ng/ml) was required for the same stimulatory
effect.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
Proliferative response of transfected BAF/3
cells to different IL-6 type cytokines. Murine BAF/3 cells were
transfected with gp130, LIFR, and CNTFR (A), gp130 and LIFR
(B), gp130 and IL-6R (C), gp130, IL-6R , and
LIFR (D) and stimulated with IL-6, LIF, CNTF, or the
recently engineered designer cytokine IC7 (55).
|
|
To further substantiate binding of CNTF to the IL-6R, we tested the
ability of the neutralizing anti-human IL-6R antibody PM-1 to block
binding of CNTF to the IL-6R. PM-1 binds to the cytokine binding
epitope of the IL-6R and thus inhibits binding of IL-6 to its cognate
-receptor (56). PM-1 did not inhibit LIF-induced proliferation of
BaF/3-[gp130,LIFR,IL-6R] cells (Fig. 2A), nor phosphorylation of
STAT3 induced by LIF (Fig. 3,
A and B). In contrast, there was a clear
competitive inhibition of BaF/3-[gp130,LIFR,IL-6R] cell proliferation
by PM-1 after stimulation with IL-6, IC7 and importantly also CNTF
(Fig. 2, B-D). Consistent with the competitive inhibition
by PM-1 of IL-6, IC7- and CNTF-dependent proliferation of
BaF/3-[gp130,LIFR,IL-6R] cells, phosphorylation of STAT3 after stimulation with CNTF was almost completely inhibited by PM-1 (Fig. 3,
A and B). Phosphorylation of STAT3 in
BaF/3-[gp130,LIFR,IL-6R] cells was also inhibited by PM-1 after
stimulation with 50 ng/ml IL-6 or IC7, but not after stimulation with
LIF. The inhibitory effect of PM-1 on CNTF-induced proliferation of
BaF/3-[gp130,LIFR,IL-6R] cells, however, was abolished when these
cells were stimulated by the combination of CNTF and the soluble CNTFR
(Fig. 2D), demonstrating that inhibition of CNTF activity by
PM-1 on BaF/3-[gp130,LIFR,IL-6R] cells was specific for interaction
of CNTF with the IL-6R.
We next wanted to analyze whether the ability of CNTF to induce
cellular responses via binding to the membrane-bound IL-6R also
pertained to the soluble IL-6R. Maximal proliferative responses of
BaF/3-[gp130,LIFR] cells to the combination IL-6/soluble IL-6R were
achieved with concentrations of sIL-6R exceeding 80 ng/ml (Fig.
5A). BaF/3-[gp130,LIFR]
cells were therefore stimulated with CNTF at concentrations of up to 1 µg/ml in the presence of 100 or 500 ng/ml sIL-6R (Fig.
5C). As in the previous experiments, maximal responses to
CNTF, which were similar to those observed after 50 ng/ml LIF (Fig.
5B), were observed at around 250 ng/ml CNTF. A marked
proliferative response was already discernible at 50 ng/ml CNTF. There
was no difference between cellular responses to CNTF in the presence of
100 and 500 ng of sIL-6R.
To demonstrate direct interaction between CNTF and the IL-6R, CNTF was
incubated with IL-6R-Fc, a recently constructed fusion protein of the
IL-6R and the Fc part of human IgG1, in the presence of 1% Brij 96 (57). Following precipitation with protein A-Sepharose and SDS-PAGE,
CNTF could be detected in the precipitate with a Western blot against
human CNTF (Fig. 6, lanes
4-7). IL-6R-Fc alone did not react in the Western blot
(lane 2). The polyclonal antibody specifically detected CNTF
(lane 1) and did not cross-react with human IL-6 (lane
3). To quantify real-time binding of IL-6 and CNTF to the IL-6R,
IL-6R-Fc was immobilized to a IASYSTM cuvette, and the
association and dissociation rates for binding of CNTF and IL-6 to
IL-6R-Fc determined (Fig. 7, A
and B). A KD of 184 nM was
measured for binding of IL-6 (kon = 3.71 × 104 M
1 s
1,
koff = 6.83 × 10
3
s
1), whereas the KD for CNTF was 9.0 µM (kon = 2.68 × 102 M
1 s
1,
koff = 2.44 × 10
3
s
1). For comparison, the reported dissociation constants
for IL-6 binding to the complete IL-6R (64) and the membrane-proximal domain of the IL-6R alone (65) are 5-fold smaller and 2-fold higher,
respectively, than the ones measured here for IL-6R-Fc. These results
suggest that the 42-fold difference in CNTF activity after binding to
the IL-6R compared with IL-6 and IC7 (Fig. 1D) most probably
reflects the roughly 50-fold lower affinity of human CNTF binding to
the human IL-6R.
To understand the difference in binding to the human IL-6R between
human and rat CNTF on a molecular level, we generated a three-dimensional model of the human CNTF/human IL-6R complex (Fig.
8A). The x-ray structure of
the human CNTF and a model of the human IL-6/ILR-6 complex were used as
templates as described under "Experimental Procedures." The human
IL-6R is bound to site I of human CNTF, and therefore we analyzed which
amino acid residues involved in the interacting epitope are conserved
in human and rat CNTF. Interestingly, we found that only one residue,
Gln-63 in human CNTF, is replaced by an arginine in the rat molecule. Fig. 8B shows a magnified view of the interaction area
between the two human molecules. Gln-63 is deeply buried in the
interface, closely surrounded by amino acid side chains from the CNTF
(Asp-62, Trp-64, His-174) and IL-6R (Pro-126, Leu-127, Tyr-188,
Ser-246, Phe-248, Gln-300). The fact that rat CNTF does not bind to the human IL-6R, therefore, results from the central involvement of the
Gln-63 side chain in the binding interface between CNTF and the IL-6R.
Compared with Gln, the side chain of arginine is substantially more
extended and can thus not be accommodated in the interaction area. The
increased size (Gln, 143.8 Å3; Arg, 173.4 Å3)
and additional charge of the arginine in rat CNTF, compared with the
Gln-63 in human CNTF, sterically prevents complex formation with the
human IL-6R. Since rat CNTF in contrast to human CNTF is able to
directly induce an active heterodimer of human gp130 and human LIFR, we
examined the structure of human CNTF and a newly created model of rat
CNTF (not shown) for differences in site II and site III that would
explain this effect. However, comparisons of these epitopes with regard
to charge distribution and hydrophobic patch extension did not reveal
an obvious cause for an increased affinity of rat CNTF site II/III to
gp130 or the LIFR.
The results presented here clearly demonstrate binding of human
CNTF to the human IL-6R with an affinity roughly 50-fold lower than
that of IL-6 binding to the IL-6R. Human CNTF is capable of eliciting a
functional gp130/LIFR heterodimer after binding to both, the
membrane-bound or the soluble IL-6R. Even at high concentrations, human
CNTF is unable to directly induce a human gp130/LIFR heterodimer.
Thus, human CNTF differs on both accounts from rat CNTF that can
directly induce an active heterodimer of human gp130 and LIFR (11) and
does not bind to the human IL-6R (52). The ability of human CNTF to
induce the acute-phase response on human liver cells, which do not
express the CNTFR (53) is therefore due to the formation of an active
receptor complex consisting of IL-6R, gp130, and LIFR (Fig. 4).
Conspicuously, although rat CNTF behaves differently than human CNTF on
human cells, it can also stimulate rat hepatocytes via the IL-6R (54).
A certain degree of caution might therefore be applied to
receptor-ligand binding studies using proteins from different species,
since the results obtained may be artifactual and reflect a
situation foreign to both species as in the case of CNTF.
The finding that the new IL-6 family member CLC, which signals via a
gp130/LIFR heterodimer, also uses the CNTFR as an
-receptor (9, 36)
already violated the concept that
-receptors primarily confer
specificity to cytokine-dependent receptor complex
assembly. Our results demonstrate that specificity of
-receptors is
not even restricted to the same subgroup of IL-6 type cytokines,
i.e. those signaling via a gp130 homo- or a gp130/LIFR
heterodimer. Specificity of
-receptors for certain cytokines may
therefore not be an absolute property of the
-receptor, but a
quantitative one. An important result of this study is that human CNTF
can induce cellular responses after binding to the soluble IL-6R. This
contrasts with the requirement of CLC for the membrane-bound CNTFR to
elicit biological responses (66). Transignaling via soluble
receptors of the IL-6 family significantly widens the array of
potential target cells (67), and recent evidence suggests that this
process plays a critical role in a variety of different diseases
(68-70).
The side effects of CNTF in the toxicity study limited the CNTF
concentration in a trial of CNTF for the treatment of amyotrophic lateral sclerosis to 5 µg/kg of body weight, where no positive effects compared with placebo were noted (51). Construction of a human
CNTF variant that specifically interacts with the human CNTFR may
improve the safety profile of CNTF and thus allow to reinvestigate
clinical benefits of the application of higher doses of the agent in
amyotrophic lateral sclerosis. In the CNTF analogue axokine, Gln-63 of
human CNTF was substituted by Arg-63 of rat CNTF (47), which is known
to increase the specific activity of human CNTF (72). Since rat CNTF
does not bind to the human IL-6R (52), it is conceivable that axokine
does not bind to the human IL-6R. Our molecular model of the human
CNTF/human IL-6R complex provides a rationale to understanding this
difference, since it shows that sterically, Arg-63 cannot be
accommodated in the interaction area between human CNTF and the human
IL-6R. Axokine may thus be both, more active and more specific than
natural human CNTF, which appears particularly desirable in view of the therapeutic potential of CNTF and may even allow the reinvestigation of
the clinical effects of CNTF signaling in ALS at higher dosages.
We thank Dr. Hugues Gascan for the polyclonal
antibody against human CNTF (INSERM, CHU Angers, France) and Werner
Meinert for technical assistance.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210044200
The abbreviations used are:
CNTF, ciliary
neurotrophic factor;
ALS, amyotrophic lateral sclerosis;
CBM, cytokine
binding module;
CLC, cardiotrphin-like cytokine;
CNTFR, CNTF receptor;
CT-1, cardiotrophin 1;
FNIII, fibronectin-type-III;
GCSF, granulocyte
colony-stimulating factor;
IL-6, interleukin-6;
IL-6R, IL-6 receptor,
IL-11, interleukin-11;
LIF, leukemia inhibitory factor;
LIFR, LIF
receptor;
MS, multiple sclerosis;
OSM, oncostatin M;
OSMR, OSM
receptor;
STAT3, signal transducer and activator of transcription 3;
MAP, mitogen-activated protein.
1.
|
Stockli, K. A.,
Lottspeich, F.,
Sendtner, M.,
Masiakowski, P.,
Carroll, P.,
Gotz, R.,
Lindholm, D.,
and Thoenen, H.
(1989)
Nature
342,
920-923[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Lin, L. F.,
Mismer, D.,
Lile, J. D.,
Armes, L. G.,
Butler, E. T. D.,
Vannice, J. L.,
and Collins, F.
(1989)
Science
246,
1023-1025[Medline]
[Order article via Infotrieve]
|
3.
|
Grötzinger, J.,
Kernebeck, T.,
Kallen, K. J.,
and Rose-John, S.
(1999)
Biol. Chem.
380,
803-813[Medline]
[Order article via Infotrieve]
|
4.
|
Shi, Y.,
Wang, W.,
Yourey, P. A.,
Gohari, S.,
Zukauskas, D.,
Zhang, J.,
Ruben, S.,
and Alderson, R. F.
(1999)
Biochem. Biophys. Res. Commun.
262,
132-138[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Senaldi, G.,
Varnum, B. C.,
Sarmiento, U.,
Starnes, C.,
Lile, J.,
Scully, S.,
Guo, J.,
Elliott, G.,
McNinch, J.,
Shaklee, C. L.,
Freeman, D.,
Manu, F.,
Simonet, W. S.,
Boone, T.,
and Chang, M. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11458-11463[Abstract/Free Full Text]
|
6.
|
Heinrich, P. C.,
Behrmann, I.,
Muller Newen, G.,
Schaper, F.,
and Graeve, L.
(1998)
Biochem. J.
334,
297-314[Medline]
[Order article via Infotrieve]
|
7.
|
Davis, S.,
Aldrich, T. H.,
Ip, N. Y.,
Stahl, N.,
Scherer, S.,
Farruggella, T.,
DiStefano, P. S.,
Curtis, R.,
Panayotatos, N.,
Gascan, H.,
Chevalier, S.,
and Yancopulos, G. D.
(1993)
Science
259,
1736-1739[Medline]
[Order article via Infotrieve]
|
8.
|
Davis, S.,
Aldrich, T. H.,
Stahl, N.,
Pan, L.,
Taga, T.,
Kishimoto, T.,
Ip, N. Y.,
and Yancopoulos, G. D.
(1993)
Science
260,
1805-1808[Medline]
[Order article via Infotrieve]
|
9.
|
Elson, G. C.,
Lelievre, E.,
Guillet, C.,
Chevalier, S.,
Plun-Favreau, H.,
Froger, J.,
Suard, I.,
de Coignac, A. B.,
Delneste, Y.,
Bonnefoy, J. Y.,
Gauchat, J. F.,
and Gascan, H.
(2000)
Nat. Neurosci.
3,
867-872[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Robledo, O.,
Fourcin, M.,
Chevalier, S.,
Guillet, C.,
Auguste, P.,
Pouplard Barthelaix, A.,
Pennica, D.,
and Gascan, H.
(1997)
J. Biol. Chem.
272,
4855-4863[Abstract/Free Full Text]
|
11.
|
Gearing, D. P.,
Ziegler, S. F.,
Comeau, M. R.,
Friend, D.,
Thoma, B.,
Cosman, D.,
Park, L.,
and Mosley, B.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1119-1123[Abstract]
|
12.
|
Mosley, B.,
De Imus, C.,
Friend, D.,
Boiani, N.,
Thoma, B.,
Park, L. S.,
and Cosman, D.
(1996)
J. Biol. Chem.
271,
32635-32643[Abstract/Free Full Text]
|
13.
|
Taga, T.,
and Kishimoto, T.
(1997)
Annu. Rev. Immunol.
15,
797-819[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Stockli, K. A.,
Lillien, L. E.,
Naher Noe, M.,
Breitfeld, G.,
Hughes, R. A.,
Raff, M. C.,
Thoenen, H.,
and Sendtner, M.
(1991)
J. Cell Biol.
115,
447-459[Abstract]
|
15.
|
Oppenheim, R. W.,
Prevette, D.,
Yin, Q. W.,
Collins, F.,
and MacDonald, J.
(1991)
Science
251,
1616-1618[Medline]
[Order article via Infotrieve]
|
16.
|
Hughes, S. M.,
Lillien, L. E.,
Raff, M. C.,
Rohrer, H.,
and Sendtner, M.
(1988)
Nature
335,
70-73[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Sendtner, M.,
Kreutzberg, G. W.,
and Thoenen, H.
(1990)
Nature
345,
440-441[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Clatterbuck, R. E.,
Price, D. L.,
and Koliatsos, V. E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
2222-2226[Abstract]
|
19.
|
Sendtner, M.,
Schmalbruch, H.,
Stockli, K. A.,
Carroll, P.,
Kreutzberg, G. W.,
and Thoenen, H.
(1992)
Nature
358,
502-504[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Anderson, K. D.,
Panayotatos, N.,
Corcoran, T. L.,
Lindsay, R. M.,
and Wiegand, S. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7346-7351[Abstract/Free Full Text]
|
21.
|
Emerich, D. F.,
Winn, S. R.,
Hantraye, P. M.,
Peschanski, M.,
Chen, E. Y.,
Chu, Y.,
McDermott, P.,
Baetge, E. E.,
and Kordower, J. H.
(1997)
Nature
386,
395-399[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
de Almeida, L. P.,
Zala, D.,
Aebischer, P.,
and Deglon, N.
(2001)
Neurobiol. Dis.
8,
433-446[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Simon, R.,
Thier, M.,
Kruttgen, A.,
Rose John, S.,
Weiergraber, O.,
Heinrich, P. C.,
Schroder, J. M.,
and Weis, J.
(1995)
Neuroreport
7,
153-157[Medline]
[Order article via Infotrieve]
|
24.
|
Larkfors, L.,
Lindsay, R. M.,
and Alderson, R. F.
(1994)
Eur. J. Neurosci.
6,
1015-1025[Medline]
[Order article via Infotrieve]
|
25.
|
Ip, N. Y.,
Li, Y. P.,
van de Stadt, I.,
Panayotatos, N.,
Alderson, R. F.,
and Lindsay, R. M.
(1991)
J. Neurosci.
11,
3124-3134[Abstract]
|
26.
|
LaVail, M. M.,
Yasumura, D.,
Matthes, M. T.,
Lau-Villacorta, C.,
Unoki, K.,
Sung, C. H.,
and Steinberg, R. H.
(1998)
Invest. Ophthalmol. Vis. Sci.
39,
592-602[Abstract]
|
27.
|
Chong, N. H.,
Alexander, R. A.,
Waters, L.,
Barnett, K. C.,
Bird, A. C.,
and Luthert, P. J.
(1999)
Invest. Ophthalmol. Vis. Sci.
40,
1298-1305[Abstract]
|
28.
|
Peterson, W. M.,
Wang, Q.,
Tzekova, R.,
and Wiegand, S. J.
(2000)
J. Neurosci.
20,
4081-4090[Abstract/Free Full Text]
|
29.
|
Helgren, M. E.,
Squinto, S. P.,
Davis, H. L.,
Parry, D. J.,
Boulton, T. G.,
Heck, C. S.,
Zhu, Y.,
Yancopoulos, G. D.,
Lindsay, R. M.,
and DiStefano, P. S.
(1994)
Cell
76,
493-504[Medline]
[Order article via Infotrieve]
|
30.
|
Guillet, C.,
Auguste, P.,
Mayo, W.,
Kreher, P.,
and Gascan, H.
(1999)
J. Neurosci.
19,
1257-1262[Abstract/Free Full Text]
|
31.
|
Masu, Y.,
Wolf, E.,
Holtmann, B.,
Sendtner, M.,
Brem, G.,
and Thoenen, H.
(1993)
Nature
365,
27-32[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Sendtner, M.,
Gotz, R.,
Holtmann, B.,
Escary, J. L.,
Masu, Y.,
Carroll, P.,
Wolf, E.,
Brem, G.,
Brulet, P.,
and Thoenen, H.
(1996)
Curr. Biol.
6,
686-694[Medline]
[Order article via Infotrieve]
|
33.
|
DeChiara, T. M.,
Vejsada, R.,
Poueymirou, W. T.,
Acheson, A.,
Suri, C.,
Conover, J. C.,
Friedman, B.,
McClain, J.,
Pan, L.,
and Stahl, N.
(1995)
Cell
83,
313-322[Medline]
[Order article via Infotrieve]
|
34.
|
Alexander, W. S.,
Rakar, S.,
Robb, L.,
Farley, A.,
Willson, T. A.,
Zhang, J. G.,
Hartley, L.,
Kikuchi, Y.,
Kojima, T.,
Nomura, H.,
Hasegawa, M.,
Maeda, M.,
Fabri, L.,
Jachno, K.,
Nash, A.,
Metcalf, D.,
Nicola, N. A.,
and Hilton, D. J.
(1999)
Curr. Biol.
9,
605-608[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Elson, G. C.,
Graber, P.,
Losberger, C.,
Herren, S.,
Gretener, D.,
Menoud, L. N.,
Wells, T. N.,
Kosco-Vilbois, M. H.,
and Gauchat, J. F.
(1998)
J. Immunol.
161,
1371-1379[Abstract/Free Full Text]
|
36.
|
Plun-Favreau, H.,
Elson, G.,
Chabbert, M.,
Froger, J.,
deLapeyriere, O.,
Lelievre, E.,
Guillet, C.,
Hermann, J.,
Gauchat, J. F.,
Gascan, H.,
and Chevalier, S.
(2001)
EMBO J.
20,
1692-1703[Abstract/Free Full Text]
|
37.
|
Takahashi, R.,
Yokoji, H.,
Misawa, H.,
Hayashi, M.,
Hu, J.,
and Deguchi, T.
(1994)
Nat. Genet.
7,
79-84[Medline]
[Order article via Infotrieve]
|
38.
|
Anand, P.,
Parrett, A.,
Martin, J.,
Zeman, S.,
Foley, P.,
Swash, M.,
Leigh, P. N.,
Cedarbaum, J. M.,
Lindsay, R. M.,
Williams Chestnut, R. E.,
and Sinicropi, D. V.
(1995)
Nat. Med.
1,
168-172[Medline]
[Order article via Infotrieve]
|
39.
|
Giess, R.,
Holtmann, B.,
Braga, M.,
Grimm, T.,
Muller-Myhsok, B.,
Toyka, K. V.,
and Sendtner, M.
(2002)
Am. J. Hum. Genet.
70,
1277-1286[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Giess, R.,
Maurer, M.,
Linker, R.,
Gold, R.,
Warmuth-Metz, M.,
Toyka, K. V.,
Sendtner, M.,
and Rieckmann, P.
(2002)
Arch. Neurol.
59,
407-409[Abstract/Free Full Text]
|
41.
|
Linker, R. A.,
Maurer, M.,
Gaupp, S.,
Martini, R.,
Holtmann, B.,
Giess, R.,
Rieckmann, P.,
Lassmann, H.,
Toyka, K. V.,
Sendtner, M.,
and Gold, R.
(2002)
Nat. Med.
8,
620-624[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Meazza, C.,
Di Marco, A.,
Fruscella, P.,
Gloaguen, I.,
Laufer, R.,
Sironi, M.,
Sipe, J. D.,
Villa, P.,
Romano, M.,
and Ghezzi, P.
(1997)
Neuroimmunomodulation
4,
271-276[Medline]
[Order article via Infotrieve]
|
43.
|
Okuda, Y.,
Sakoda, S.,
Bernard, C. C.,
Fujimura, H.,
Saeki, Y.,
Kishimoto, T.,
and Yanagihara, T.
(1998)
Int. Immunol.
10,
703-708[Abstract]
|
44.
|
Eugster, H. P.,
Frei, K.,
Kopf, M.,
Lassmann, H.,
and Fontana, A.
(1998)
Eur. J. Immunol.
28,
2178-2187[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Mendel, I.,
Katz, A.,
Kozak, N.,
Ben Nun, A.,
and Revel, M.
(1998)
Eur. J. Immunol.
28,
1727-1737[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Samoilova, E. B.,
Horton, J. L.,
Hilliard, B.,
Liu, T. S.,
and Chen, Y.
(1998)
J. Immunol.
161,
6480-6486[Abstract/Free Full Text]
|
47.
|
Lambert, P. D.,
Anderson, K. D.,
Sleeman, M. W.,
Wong, V.,
Tan, J.,
Hijarunguru, A.,
Corcoran, T. L.,
Murray, J. D.,
Thabet, K. E.,
Yancopoulos, G. D.,
and Wiegand, S. J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4652-4657[Abstract/Free Full Text]
|
48.
|
Gloaguen, I.,
Costa, P.,
Demartis, A.,
Lazzaro, D.,
Di Marco, A.,
Graziani, R.,
Paonessa, G.,
Chen, F.,
Rosenblum, C. I.,
Van der Ploeg, L. H.,
Cortese, R.,
Ciliberto, G.,
and Laufer, R.
(1997)
Proc. Natl. Acad. Sci.
94,
6456-6461[Abstract/Free Full Text]
|
49.
|
Kalra, S. P.
(2001)
Proc. Natl. Acad. Sci.
98,
4279-4281[Free Full Text]
|
50.
|
Miller, R. G.,
Bryan, W. W.,
Dietz, M. A.,
Munsat, T. L.,
Petajan, J. H.,
Smith, S. A.,
and Goodpasture, J. C.
(1996)
Neurology
47,
1329-1331[Abstract]
|
51.
|
Miller, R. G.,
Petajan, J. H.,
Bryan, W. W.,
Armon, C.,
Barohn, R. J.,
Goodpasture, J. C.,
Hoagland, R. J.,
Parry, G. J.,
Ross, M. A.,
and Stromatt, S. C.
(1996)
Ann. Neurol.
39,
256-260[Medline]
[Order article via Infotrieve]
|
52.
|
Schooltink, H.,
Stoyan, T.,
Roeb, E.,
Heinrich, P. C.,
and Rose-John, S.
(1992)
FEBS Lett.
314,
280-284[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Baumann, H.,
Ziegler, S. F.,
Mosley, B.,
Morella, K. K.,
Pajovic, S.,
and Gearing, D. P.
(1993)
J. Biol. Chem.
268,
8414-8417[Abstract/Free Full Text]
|
54.
|
Nesbitt, J. E.,
Fuentes, N. L.,
and Fuller, G. M.
(1993)
Biochem. Biophys. Res. Commun.
190,
544-550[CrossRef][Medline]
[Order article via Infotrieve]
|
55.
|
Kallen, K.-J.,
Grötzinger, J.,
Lelièvre, E.,
Vollmer, P.,
Aasland, D.,
Renné, C.,
Müllberg, J.,
Meyer zum Büschenfelde, K.-H.,
Gascan, H.,
and Rose-John, S.
(1999)
J. Biol. Chem.
274,
11859-11867[Abstract/Free Full Text]
|
56.
|
Sato, K.,
Tsuchiya, M.,
Saldanha, J.,
Koishihara, Y.,
Ohsugi, Y.,
Kishimoto, T.,
and Bendig, M. M.
(1993)
Cancer Res.
53,
851-856[Abstract]
|
57.
|
Jostock, T.,
Blinn, G.,
Renné, C.,
Kallen, K.-J.,
Rose-John, S.,
and Müllberg, J.
(1998)
J. Immunol. Methods
273,
173-181
|
58.
|
Aasland, D.,
Oppmann, B.,
Grötzinger, J.,
Rose-John, S.,
and Kallen, K.-J.
(2002)
J. Mol. Biol.
315,
637-646[CrossRef][Medline]
[Order article via Infotrieve]
|
59.
|
McDonald, N. Q.,
Panayotatos, N.,
and Hendrickson, W. A.
(1995)
EMBO J.
14,
2689-2699[Abstract]
|
60.
|
Grötzinger, J.,
Kurapkat, G.,
Wollmer, A.,
Kalai, M.,
and Rose John, S.
(1997)
Proteins
27,
96-109[CrossRef][Medline]
[Order article via Infotrieve]
|
61.
|
van Gunsteren, W. F.,
Billeter, S. R.,
Eising, A. A.,
Hünenberger, P. H.,
Krüger, P.,
Mark, A. E.,
Scott, W. R. P.,
and Tironi, I. G.
(1996)
Biomolecular Simulation: The GROMOS96 Manual and User Guide
, vdf Hochschulverlag AG, Zürich
|
62.
|
Vriend, G.
(1990)
J. Mol. Graph.
8,
52-56[CrossRef][Medline]
[Order article via Infotrieve]
|
63.
|
Carson, M.
(1991)
J. Appl. Crystallogr.
24,
946-950[CrossRef]
|
64.
|
Weiergräber, O.,
Hemmann, U.,
Kuster, A.,
Müller-Newen, G.,
Schneider, J.,
Rose-John, S.,
Kurschat, P.,
Brakenhoff, J. P.,
Hart, M. H.,
Stabel, S.,
and Heinrich, P.
(1995)
Eur. J. Biochem.
234,
661-669[Abstract]
|
65.
|
Özbek, S.,
Grötzinger, J.,
Krebs, B.,
Fischer, M.,
Wollmer, A.,
Jostock, T.,
Mullberg, J.,
and Rose John, S.
(1998)
J. Biol. Chem.
273,
21374-21379[Abstract/Free Full Text]
|
66.
|
Lelievre, E.,
Plun-Favreau, H.,
Chevalier, S.,
Froger, J.,
Guillet, C.,
Elson, G. C.,
Gauchat, J. F.,
and Gascan, H.
(2001)
J. Biol. Chem.
276,
22476-22484[Abstract/Free Full Text]
|
67.
|
Rose-John, S.,
and Heinrich, P. C.
(1994)
Biochem. J.
300,
281-290[Medline]
[Order article via Infotrieve]
|
68.
|
Atreya, R.,
Mudter, J.,
Finotto, S.,
Müllberg, J.,
Jostock, T.,
Wirtz, S.,
Schütz, M.,
Bartsch, B.,
Holtmann, M.,
Becker, C.,
Strand, D.,
Czaja, J.,
Schlaak, J. F.,
Lehr, H. A.,
Autschbach, F.,
Schürmann, G.,
Nishimoto, N.,
Yoshizaki, K.,
Ito, H.,
Kishimoto, T.,
Galle, P. R.,
Rose-John, S.,
and Neurath, M. F.
(2000)
Nat. Med.
6,
583-588[CrossRef][Medline]
[Order article via Infotrieve]
|
69.
|
Hurst, S. M.,
Wilkinson, T. S.,
McLoughlin, R. M.,
Jones, S.,
Horiuchi, S.,
Yamamoto, N.,
Rose-John, S.,
Fuller, G. M.,
Topley, N.,
and Jones, S. A.
(2001)
Immunity
14,
705-714[CrossRef][Medline]
[Order article via Infotrieve]
|
70.
|
Kallen, K.-J.
(2002)
Biochim. Biophys. Acta
1592,
323-343[Medline]
[Order article via Infotrieve]
|
71.
|
Robak, T.,
Gladalska, A.,
Stepien, H.,
and Robak, E.
(1998)
Mediators Inflamm.
7,
347-353[CrossRef][Medline]
[Order article via Infotrieve]
|
72.
|
Panayotatos, N.,
Radziejewska, E.,
Acheson, A.,
Pearsall, D.,
Thadani, A.,
and Wong, V.
(1993)
J. Biol. Chem.
268,
19000-19003[Abstract/Free Full Text]
|
73.
|
Chen, G. C.,
and Yang, J. T.
(1977)
Anal. Lett.
10,
1195-1207
|