Tài liệu Báo cáo Y học: Differential response of neuronal cells to a fusion protein of ciliary neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory factor pot

Differential response of neuronal cells to a fusion protein of ciliary
neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory
factor
Pia Ma¨rz
1,
*, Suat O
¨
zbek
2,
*, Martina Fischer
3
, Nicole Voltz
4
, Uwe Otten
1
and Stefan Rose-John
4,5
1
Department of Physiology, University of Basel, Switzerland;
2
Department of Biophysical Chemistry, Biocenter, University of Basel,
Switzerland;
3
Xerion Pharmaceuticals, Martinsried, Germany;
4
Department of Medicine, Section Pathophysiology, Johannes
Gutenberg University of Mainz, Germany;
5
Department of Biochemistry, Christian Albrechts University of Kiel, Germany
Ciliary neurotrophic factor (CNTF) displays neurotrophic
activities on motor neurons and neural cell populations both

in vivo and in vitro. On target cells lacking intrinsic expression
of specific receptor a subunits cytokines of the IL-6 family
only act in the presence of their specific agonistic soluble
receptors. Here, we report the construction and expression of
a CNTF/soluble CNTF-receptor (sCNTF-R) fusion protein
(Hyper-CNTF) with enhanced biological activity on cells
expressing gp130 and leukemia inhibitory factor receptor
(LIF-R), but not membrane-bound CNTF-R. At the cDNA
level, the C-terminus of the extracellular domain of human
CNTF-R (amino acids 1–346) was linked via a single glycine
residue to the N-terminus of human CNTF (amino acids
1–186). Recombinant Hyper-CNTF protein was expressed
in COS-7 cells. Hyper-CNTF efficiently induced dose-
dependent STAT3 phosphorylation and proliferation of
BAF-3 cells stably transfected with gp130 and LIF-R
cDNAs. While on BAF3/gp130/LIF-R cells, Hyper-CNTF
and LIF exhibited similar biological responses, the activity
of Hyper-CNTF on pheochromocytoma cells (PC12 cells)
was quite distinct from that of LIF. In contrast to LIF,
Hyper-CNTF stimulated neurite outgrowth of PC12 cells in
a time- and dose-dependent manner correlating with the
ability to phosphorylate MAP kinases. These data indicate
that although LIF and Hyper-CNTF use the same
heterodimeric receptor complex of gp130 and LIFR, only
Hyper-CNTF induces neuronal differentiation. The thera-
peutic potential of Hyper-CNTF as a superagonistic
neurotrophin is discussed.
Keywords: cytokines; differentiation; rat; PC12 cells; signal
transduction.
Ciliary neurotrophic factor (CNTF) is a survival and

differentiation factor for a variety of neuronal and glial cells.
It has been proposed to act as a lesion factor preventing
motor neuron degeneration after injury [1] and exerting
myotrophic activity on denervated skeletal muscle [2].
CNTF belongs to the IL-6 type family of neuropoietic
cytokines that comprises interleukin-6 (IL-6), interleukin-11
(IL-11), leukemia inhibitory factor (LIF), oncostatin M,
cardiotrophin-1 (CT-1), and novel neurotrophin-1 (NNT-
1)/cardiotrophin-like cytokine (CLC) [3–7]. All IL-6 type
cytokines use a membrane spanning 130-kDa glycoprotein,
gp130, as a signal transducing receptor subunit. The
biological response to CNTF is elicited by formation of a
multimeric receptor complex [8]. CNTF first binds
to a specific glycosyl-phosphatidylinositol-anchored a unit,
CNTF receptor (CNTF-R), which is not involved in
signaling. This is followed by the recruitment of gp130
and LIF receptor (LIF-R) as signal transducing b units,
which in turn form a disulfide-linked heterodimer that
activates the JAK/STAT and the Ras/MAP kinase path-
ways [6,9]. IL-6, CNTF as well as IL-11 and presumably
CT-1 and NNT-1 act via specific membrane receptors which
together with their ligands associate with signal transducing
b subunits thereby initiating cytoplasmic signaling. Cells
that only express signal transducing but no ligand binding
subunits for these cytokines are refractory to stimulation.
An unusual feature of the IL-6 cytokine family is that the
soluble forms of the ligand binding receptor subunits
generated by one cell type in complex with their ligands can
directly stimulate the signal transducing receptor b subunits
on different cell types which lack ligand binding a subunits

[10]. This process has been named trans-signaling [11,12].
The soluble form of CNTF-R (sCNTF-R) can be
produced by limited proteolysis or by phospholipase
C-mediated cleavage [13]. Evidence for the importance of
soluble cytokine receptors in neuronal signaling, differenti-
ation and survival responses has accumulated (reviewed in
[14]).
Most recently, it was shown that the CNTF-R is also the
cellular receptor for an additional cytokine, cardiotrophin-
like cytokine (CLC) [15]. This fact explains the different
phenotype of CNTF
–/–
and CNTF-R
–/–
mice. Whereas
CNTF
–/–
mice show a mild phenotype [16] CNTF-R
–/–
mice
die shortly after birth [17].
Correspondence to P. Ma
¨
rz, Institute of Physiology,
University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland,
Fax: + 41 61 267 3559, Tel.: + 41 61 267 3553,
E-mail:
Abbreviations: CNTF, ciliary neurotrophic factor; sCNTF-R, soluble
CNTF receptor; IL-6, interleukin-6; LIF, leukemia inhibitory factor;
CT-1, cardiotrophin-1; NNT-1, novel neurotrophin-1; CLC,

cardiotrophin-like cytokine; JAK, Janus kinase; STAT, signal
transducer and activator of transcription; MAPK, mitogen activated
protein kinase; DMEM, Dulbecco’s modified Eagle’s medium.
*Note: these authors contributed equally to this work.
(Received 6 February 2002, revised 25 April 2002, accepted 3 May 2002)
Eur. J. Biochem. 269, 3023–3031 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02977.x
Superagonistic cytokines have been designed that consist
of covalently linked cytokines and soluble receptors. The
first such molecule was Hyper-IL-6, a fusion protein in
which IL-6 and soluble IL-6-R were connected by a flexible
polypeptide linker. Hyper-IL-6 turned out to be fully active
on cells expressing gp130 at 100–1000 fold lower concen-
trations than unlinked IL-6 and sIL-6R [18]. This approach
has been adopted to obtain a superagonist of IL-11 and sIL-
11R [19].
We generated a CNTF/soluble CNTF-receptor (sCNTF-
R) fusion protein with superagonistic activity on target cells
expressing gp130 and LIF-R, but lacking membrane-bound
CNTF-R. In contrast to the existing cytokine–cytokine
receptor fusion proteins, Hyper-IL-6 and Hyper-IL-11,
which directly activate the ubiquitously expressed gp130
protein, such a protein allows more specificity due to the
restricted expression pattern of the LIF-R. While the effects
of Hyper-CNTF and LIF on BAF3/-gp130/LIF-R cells
were similar, Hyper-CNTF but not LIF induced neuronal
differentiation of rat pheochromocytoma cells (PC12).
These data point to a cell-specific difference in signaling
via the heterodimeric receptor complex of gp130 and LIF-R.
MATERIALS AND METHODS
Chemicals

Dulbecco’s modified Eagle’s medium, penicillin and strepto-
mycin were purchased from Gibco (Eggenstein, Germany).
Fetal bovine serum was obtained from Seromed (Berlin,
Germany). DEAE-dextran was purchased from Sigma
(Taufkirchen, Germany). Restriction enzymes were from
New England Biolabs (Schwalbach, Germany). T4-DNA
ligase and polynucleotide kinase were purchased from
Boehringer Mannheim (Mannheim, Germany). Protein A
Sepharose was obtained from Pharmacia (Freiburg,
Germany). Tran-
35
S-Label (44 TBqÆmmol
)1
)wasfrom
ICN (Meckenheim, Germany) and [
3
H]thymidine (74
GBqÆmmol
)1
) was obtained from Amersham International
(Aylesbury, UK). X-ray films (X-OMAT-AR) were from
Eastman Kodak (Rochester, NJ).
Cells, cytokines and antibodies
PC12 and COS-7 cells (ATCC, Manassas, VA, USA),
BAF/3-gp130 cells (Immunex, Seattle, WA, USA) and
BAF/3-gp130/LIF-R cells [20] were grown in DMEM
with glutamax (Life Technologies, Inc., Karlsruhe,
Germany), supplemented with penicillin (50 UÆmL
)1
),

streptomycin (50 lgÆmL
)1
), and 10% fetal bovine serum
at 5% CO
2
in a water saturated atmosphere. BAF/3-
gp130 cells were cultured in the presence of 10 ngÆmL
)1
Hyper-IL-6, BAF/3-gp130/LIF-R cells with 5 ngÆmL
)1
human LIF. Recombinant human IL-6 and human
CNTF were prepared as described previously [21,22].
The fusion protein hIL-6/hsIL-6R designated Hyper-IL-6
was expressed in the methylotrophic yeast Pichia pastoris
and purified to homogeneity by ion-exchange chromatog-
raphy followed by gelfiltration as described previously
[18,23]. Nerve growth factor (NGF) was isolated [24] with
modifications as described previously [25]. Recombinant
human LIF was expressed as glutathione S-transferase
(GST)-fusion protein, purified by glutathione Sepharose
4B and cleaved from GST by thrombin treatment as
described by the manufacturer (Pharmacia, Freiburg,
Germany). The fusion proteins gp130-Fc and LIF-R-Fc
were transiently expressed in COS-7 cells and purified by
protein A-Sepharose, as described previously [26,27].
Recombinant growth factor concentrations were estimated
using standard protein assays. The polyclonal anti-(phos-
pho-STAT3) Ig and anti-(phospho-p44/42 MAP kinase)
Ig were from New England Biolabs (Schwalbach,
Germany). The monoclonal anti-(CNTF-R) Ig (AN-D3)

was a kind gift of H. Gascan (Angers, France) [28].
Construction of Hyper-CNTF expression plasmid
The cDNA sequences of human CNTF-R encoding the
Ig-like domain and the cytokine binding domains (cor-
responding to amino acids 1–346) and human CNTF
(corresponding to amino acids 1–186) were amplified by
standard PCR technique. Using oligonucleotide primers,
XbaIandSmaI restriction sites were introduced at the 5¢
and 3¢ ends of the CNTF and CNTF-R cDNAs,
respectively. The primer sequences used are available
from the authors upon request. After digestion, both PCR
products were ligated simultaneously into the XbaIsiteof
the pcDNA3.1(–) expression vector (Invitrogen, San
Diego, CA, USA). Ligation at SmaI led to the insertion
of three additional nucleotides coding for glycine. The
integrity of the construct was verified by restriction
fragment analysis and DNA sequencing according to
standard protocols [29].
Expression of Hyper-CNTF in COS-7 cells
COS-7 cells were transiently transfected with plasmids
coding for either Hyper-CNTF or b-galactosidase as control
by the DEAE-Dextran technique, essentially as described
previously [30]. For immunoprecipitations, Hyper-CNTF
transfected cells were cultured for 48 h and metabolically
labeled with 50 lCiÆmL
)1
[
35
S]methionine/[
35

S]cysteine
(Tran-
35
S-Label) in methionine/cysteine-free medium for
6 h. For production of Hyper-CNTF protein, transfected
cells were transferred to serum-free medium after 24 h and
supernatants were collected on day 4 post-transfection.
Immunoprecipitation
Metabolically labeled Hyper-CNTF was precipitated from
culture media using 0.5 lgÆmL
)1
gp130-Fc, 0.5 lgÆmL
)1
LIF-R-Fc or 1 lgÆmL
)1
monoclonal anti-CNTF-R Ig
(AN-D3) followed by protein A–Sepharose. Immune
complexes were analyzed by SDS/PAGE [31] and visual-
ized by fluorography using the fluorographic intensifier
solution ÔAmplifyÕ (Amersham International, Aylesbury,
UK).
Proliferation assays
BAF/gp130 and BAF/gp130/LIF-R cells were extensively
washed with NaCl/P
i
, and resuspended in cytokine free
medium at 5 · 10
3
cells per well of a 96-well plate. They
were cultured in a final volume of 100 lL with cytokines as

indicated in the figure legends for 68 h and subsequently
pulse labeled with 0.25 lCi [
3
H]thymidine for 4 h. Cells
3024 P. Ma
¨
rz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
were harvested on glass filters and incorporated [
3
H]
thymidine was determined by scintillation counting. Inde-
pendent bioassays were performed three times with each
value being determined in duplicate.
Neurite outgrowth
Neurite outgrowth assays were performed in six-well plates.
PC12 cells were grown in complete media in the presence of
growth factors as indicated. The percentage of responsive
cells characterized by neurites extending longer than twice
the diameter of cell bodies was scored. The scale of
microphotographs is indicated in the figure legends as fold
magnification.
Western Blot analysis
Proteins from cell lysates of transfected BAF/3 or PC12 cells
were separated by SDS/PAGE and transferred onto
poly(vinylidine fluoride) membranes by electroblotting.
Phosphorylated STAT3 and p44/42 MAP kinases (New
England Biolabs, Schwalbach, Germany), were detected
using polyclonal rabbit anti-(phospho-STAT3) Ig and anti-
(phospho-p44/42 MAP kinase) Ig. As secondary reagent,
horseradish peroxidase (HRP)-conjugated goat anti-(rabbit

IgG) Ig was used (Sigma, Deisenhofen, Germany). The blot
was developed using the ECL-detection system (Amersham
International, Aylesbury, UK). The STAT3 and MAPK
phosphorylation assays were reproduced three times with
one representative experiment shown.
RESULTS
Construction of CNTF/sCNTF-R fusion protein
We engineered an expression vector encoding a CNTF/
sCNTF-R fusion protein by linking the C-terminus of
human CNTF-R to the N-terminus of human CNTF
(Fig. 1A). In principle, we followed the design of Hyper-
IL-6 [18] with two specific modifications. First, we included
the N-terminal Ig domain of the sCNTF-R, as deletion of
this region lead to reduced expression levels of recombinant
sCNTF-RDIg protein (P. Ma
¨
rz, M. Fischer & S. Rose-
John, unpublished work). This observation is in line with
recent results indicating that the Ig-like domain of the IL-6R
is important for intracellular transport of IL-6R through the
secretory pathway [32]. Secondly, we avoided the use of a
synthetic polypeptide linker in order to minimize immun-
ogenicity. Instead, the 16 C-terminal amino acids of
CNTF-R (amino acids 331–346) that are not part of the
membrane-proximal cytokine binding domain [33] and the
14 N-terminal nonhelical and presumably flexible amino
acids of CNTF (amino acids 1–12) [34] were linked by one
additional glycine residue. The resulting length of 31 amino
acids, in analogy to Hyper-IL-6 and Hyper-IL-11, is
presumably sufficient to connect both molecules and to

allow access of CNTF to its CNTF-R binding site. In a
similar approach, we have recently reduced the length of the
Hyper-IL-6 linker without apparent loss of biological
function [35]. A schematic model of the anticipated tertiary
structure of the CNTF/sCNTF-R fusion protein is shown in
Fig. 1B.
Expression of CNTF/sCNTF-R fusion protein
and interaction with the signal transducing
b-subunits gp130 and LIF-R
Expression of Hyper-CNTF protein was performed by
transient transfection of COS-7 cells. Cleavage of the
endogenous CNTF-R signal peptide in transfected COS
cells led to the secretion of the fusion protein Hyper-CNTF
into the supernatant. As shown in Fig. 2A, the Hyper-
CNTF fusion protein, with an apparent molecular mass of
 82 kDa, was detected by Western blot analysis with a
CNTF antiserum. Supernatant from mock transfected
COS-7 cells expressing the b-Gal gene did not show any
signal detected by the CNTF antiserum. Immunodetection
with this antibody also serves as control for complete
translation and integrity of Hyper-CNTF protein because it
recognizes the C-terminal CNTF moiety of the newly
generated protein. After transfection of COS-7 cells with the
Hyper-CNTF expression plasmid, metabolically
35
S-labeled
Hyper-CNTF protein could be precipitated from the super-
natant with a monoclonal anti-(CNTF-R) Ig (Fig. 2B). To
test for physical interaction with the signal transducing
b subunits of the CNTF-R system, the

35
S-labeled Hyper-
CNTF protein was incubated with Fc-fusion proteins
containing the extracellular portion of gp130 or the
extracellular portion of LIF-R. Protein complexes were
precipitated with protein A-Sepharose. As can be seen
in Fig. 2B, Hyper-CNTF interacted with gp130-Fc and
LIF-R-Fc to a similar extent.
Fig. 1. Schematic representation of the fusion protein of CNTF and
sCNTF-R. (A) Construction of the fusion protein. The C-terminus of
sCNTF-R was linked via one additional glycine residue (G) to the
N-terminus of CNTF. (B) Schematic model of the Hyper-CNTF ter-
tiary structure. Ig denotes the immunoglobulin-like domain, D2 and
D3 the two cytokine-binding receptor domains.
Ó FEBS 2002 CNTF/sCNTF-R fusion protein with enhanced activity (Eur. J. Biochem. 269) 3025
Biological activity of the Hyper-CNTF fusion protein
To assess the biological activity of Hyper-CNTF, we first
investigated the proliferative response of transfected BAF/3
cells. Murine BAF/3 cells, which normally grow IL-3-
dependently, are known to proliferate in response to various
cytokines after transfection of the corresponding receptor
chains. BAF/3 cells transfected with human gp130 and/or
additional transfection of the human LIF-R were stimulated
with increasing amounts of Hyper-IL-6, Hyper-CNTF, LIF
or medium alone. Proliferation of cells was assayed by
measuring [
3
H]thymidine incorporation into DNA. As
shown in Fig. 3A, BAF/3-gp130 cells proliferate upon
stimulation with Hyper-IL-6, but absence of the LIF-R

prevented a proliferative activity of LIF as well as of Hyper-
CNTF on these cells. In contrast, on BAF/3-gp130/LIF-R
cells Hyper-IL-6, LIF and Hyper-CNTF were fully active
(Fig. 3B). These data indicate that fusion of CNTF to its
respective soluble CNTF-R resulted in a protein conferring
responsiveness of cells that lack membrane-bound CNTF-R
and thus are usually inert to stimulation by CNTF. The
most significant finding, however, is that Hyper-CNTF has
virtually the same activity as LIF as well as Hyper-IL-6;
half-maximal activity was obtained with cytokine concen-
trations of 5–10 pgÆmL
)1
. Accordingly, Hyper-CNTF rep-
resents a protein with greatly enhanced bioactivity requiring
heterodimerization of the b-receptor subunits gp130 and
LIF-R.
Fig. 2. Interaction of the Hyper-CNTF protein with the signaling
receptor subunits gp130 and LIF-R. (A) Immunodetection of Hyper-
CNTF protein in the supernatants of transiently transfected COS-7
cells. The C-terminal CNTF moiety of the fusion protein was detected
with a polyclonal CNTF antiserum [22]. Recombinant human CNTF
(at 26 kDa) was blotted as positive control and supernatant from
mock transfected COS-7 cells expressing the b-gal gene served as
negative control. (B) Metabolically labeled Hyper-CNTF was preci-
pitated from cell supernatants with gp130-Fc, LIFR-Fc proteins or a
monoclonal anti-(CNTF-R) Ig. Immune complexes precipitated with
protein A–Sepharose were separated by SDS/PAGE and visualized by
fluorography. Electrophoretic mobilities of molecular mass marker
proteins are indicated on the left.
Fig. 3. Proliferative response of transfected BAF/3 cells to Hyper-

CNTF. (A) BAF/3-gp130 cells and (B) BAF/3-gp130/LIF-R cells were
stimulated with increasing amounts of Hyper-CNTF, Hyper-IL-6, LIF
or medium alone. Proliferation of cells was assayed by measuring
[
3
H]thymidine incorporation into DNA. One representative experi-
ment is shown.
3026 P. Ma
¨
rz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
STAT3 and MAPK activation by Hyper-CNTF
in transfected BAF/3 cells
Downstream signal transduction pathways were analyzed
by studying the activation level of JAK/STAT and MAP
kinase signaling components known to be mainly tyrosine
phosphorylated in response to IL-6 type cytokines [36–38].
BAF/3-gp130 cells and BAF/3-gp130/LIF-R cells were
stimulated with medium alone, 10 ngÆmL
)1
Hyper-IL-6,
20 ngÆmL
)1
Hyper-CNTF, 50 ngÆmL
)1
IL-6, 50 ngÆmL
)1
CNTF or 20 ngÆmL
)1
LIF for 10 min (Fig. 4). Cells were
lysed in Laemmli buffer and proteins were separated via

SDS/PAGE and blotted onto poly(vinylidine fluoride)
membranes. Membranes were cut into two pieces below
the 62-kDa marker band and phosphorylated STAT3
proteins were detected on the upper part of the membrane
using a phosphospecific anti-STAT3 Ig. Analogously,
phosphorylated MAP kinases were detected on the lower
part of the membrane by use of a phospho-p44/42 MAP
kinase antibody followed by ECL detection. As shown in
Fig. 4, a 10-min stimulation of BAF/3-gp130 cells with
Hyper-IL-6 led to pronounced tyrosine phosphorylation of
STAT3 and p42/p44 MAP kinases. The same activation
pattern was observed after stimulation of BAF/3-gp130/
LIF-R cells with Hyper-IL-6, Hyper-CNTF and LIF. No
phosphorylation could be detected upon stimulation of the
cells with CNTF or IL-6, reflecting the lack of the specific
ligand binding receptor subunits or medium alone in none
of the two transfected BAF/3 cell lines. These data indicate
that on BAF/3 cells, the composite cytokines Hyper-IL-6
and Hyper-CNTF as well as LIF recruit the same signal
transduction pathways for induction of proliferation with-
out any receptor-specific differences.
Neuronal differentiation of PC12 cells by Hyper-CNTF
In a second bioassay, we investigated the potential role
of Hyper-CNTF in neuronal cell differentiation. The
morphology of rat pheochromocytoma cells (PC12) grown
for 48 h in serum-containing medium in the absence of
factors (medium) or in the presence of 100 ngÆmL
)1
CNTF,
20 ngÆmL

)1
Hyper-CNTF, 100 ngÆmL
)1
LIF, 100 ngÆmL
)1
NGF or 20 ngÆmL
)1
Hyper-IL-6 was analysed. As expected
from earlier studies [39,40], stimulation of the cells with
NGF or Hyper-IL-6 led to robust formation of neurites.
Surprisingly, exposure of the cells to Hyper-CNTF also
induced pronounced neuronal differentiation, whereas LIF
and CNTF (at concentrations up to 500 ngÆmL
)1
, data not
shown) did not result in significant morphological changes
(Fig. 5A). Hyper-CNTF induced neurites extending longer
than twice the diameter of the cell bodies appear within a
day, and maximal response is reached in 2 days. For direct
comparison, the amount of responsiveness was evaluated
forallfactorsat48h.AspresentedinFig.5B,Hyper-
CNTF turned out to be virtually as effective as NGF and
Hyper-IL-6 to elicit neuronal differentiation.
STAT3 and MAPK activation by Hyper-CNTF in PC12 cells
We then asked which signal transduction pathways are
involved in Hyper-CNTF-induced neurite outgrowth. In a
first experiment, PC12 cells were stimulated with medium
alone, Hyper-IL-6, NGF, Hyper-CNTF, or LIF for 10 min
(Fig. 6A). Cells were lysed in Laemmli buffer and cell lysates
Fig. 4. STAT3 and MAPK phosphorylation by Hyper-CNTF in

transfected BAF/3 cells. (A) BAF/3-gp130 cells and (B) BAF/3-gp130/
LIF-R cells were stimulated with medium alone, 10 ngÆmL
)1
Hyper-
IL-6, 20 ngÆmL
)1
Hyper-CNTF, 50 ngÆmL
)1
IL-6, 50 ngÆmL
)1
CNTF
or 20 ngÆmL
)1
LIF for 10 min. Cells were lysed in Laemmli buffer and
proteins were separated via SDS/PAGE and blotted onto PVDF
membranes. Membranes were probed for phosphorylated STAT3 and
MAP kinases (p44/p42) using phospho-specific antibodies and ECL
detection.
Fig. 5. Neuronal differentiation of PC12 cells by Hyper-CNTF. (A)
Morphology of PC12 cells grown for 48 h in serum-containing
medium in the absence of factors (medium) or in the presence of
100 ngÆmL
)1
CNTF, 20 ngÆmL
)1
Hyper-CNTF, 100 ngÆmL
)1
LIF,
100 ngÆmL
)1

NGF or 20 ngÆmL
)1
Hyper-IL-6 was analyzed (magni-
fication: 300·) and (B) the extent of responsiveness was evaluated by
analysis of neurite outgrowth. Vertical bars represent S.E.M. (n ¼ 3).
Ó FEBS 2002 CNTF/sCNTF-R fusion protein with enhanced activity (Eur. J. Biochem. 269) 3027
were analyzed for STAT3 and MAPK phosphorylation as
described above. We found that stimulation with Hyper-IL-
6 led to an increase of both, STAT3 and MAPK phos-
phorylation. Consistent with other reports [41,42], a strong
activation of p42/p44 MAP kinases was observed by NGF.
Interestingly, as compared to Hyper-IL-6, treatment of the
cells with Hyper-CNTF resulted in small but significant
STAT3 phosphorylation and strong MAPK phosphoryla-
tion which was at least equal to Hyper-IL-6. In contrast,
stimulation with LIF alone had a similar effect on STAT3
phosphorylation but no effect on MAP kinase activation. As
demonstrated in Fig. 6B, the dose–response phosphoryla-
tion pattern for both STAT3 and p42/p44 MAP kinases
clearly confirmed that of the cytokines signaling through
gp130/LIF-R only Hyper-CNTF but not LIF or CNTF
(even at high concentrations) alone were able to activate the
MAPK pathway. MAP kinases and STAT3 are rapidly
activated within 10 min in response to Hyper-CNTF, the
phase of activation lasting for at least 30 min before
returning to near basal levels within 1 h (Fig. 6C). These
data are in line with the different abilities of Hyper-CNTF,
LIF and CNTF to induce neuronal differentiation in PC12
cells, as observed above. We conclude that the Hyper-
CNTF-induced neurite outgrowth is most likely mediated by

activation of the MAPK pathway and that this response is
substantially independent of the JAK/STAT pathway.
DISCUSSION
We have successfully expressed an active fusion protein of
human CNTF and human soluble CNTF-R in mammalian
cells. Hyper-CNTF has a calculated molecular mass of
60 kDa and apparent molecular mass of 85 kDa, the
increase being most likely due to heavy glycosylation (four
N-linked glycosylation sites). Expression of Hyper-CNTF
circumvents the use of high amounts of recombinant CNTF
and soluble CNTF-R since the concentrations of the two
separate components needed for full stimulation is 1–2
orders of magnitude higher than that of Hyper-CNTF [13].
The Hyper-CNTF protein was precipitated using Fc
fusion proteins of the extracellular portion of gp130 and
LIF-R [26]. This result confirms the structural integrity of
the Hyper-CNTF protein since the CNTF/sCNTF-R
complex has been reported to interact with the LIF-R.
Direct binding of the CNTF/sCNTF-R complex to gp130
has not been previously described. It has been noted,
however, that LIF bound not only to the LIF-R but also to
the gp130 protein albeit with low affinity [43–45].
Cells that only express gp130 and LIF-R, but not CNTF-
Ra are refractory to stimulation by CNTF. As expected,
BAF/3-gp130 cells lacking LIF-R were neither responsive to
Hyper-CNTF nor to LIF. Hyper-CNTF induced prolifer-
ation of BAF/3 cells expressing gp130 and LIF-R at
virtually the same concentration as LIF and Hyper-IL-6
needed to achieve half-maximal activity. The signaling
events of stimulated BAF/3 cells reflected by the activation

pattern of STAT3 and MAP kinases, mainly p42, were
identical for BAF/3 cells stimulated with Hyper-IL-6,
Hyper-CNTF, and LIF.
Analysis of the biological activity of Hyper-CNTF in
non-neuronal vs. neuronal cells revealed unexpected func-
tional and biochemical differences between LIF and Hyper-
CNTF activity. In contrast to LIF, Hyper-CNTF rapidly
induced neurite outgrowth and formation of a neuronal
network in PC12 cells. Looking at the signaling events, we
observed that both LIF and Hyper-CNTF induced phos-
phorylation of STAT3. However, only Hyper-CNTF has
the potential to activate MAP kinases. This finding is in
agreement with the experiments of Sterneck et al. who failed
to induce neuronal differentiation with CNTF and LIF in
PC12 cells [46,47].
How can the differential response of BAF/3 cells and
PC12 cells be explained? The phenomenon that stimulation
of the gp130/LIF-R complex by different cytokines might
result in different biological responses in neuronal cells has
already been discussed in a review [48]. It is known that
gp130 stimulation leads to the activation of multiple
signaling cascades including the STAT3 and the MAPK
Fig. 6. STAT3 and MAPK activation by Hyper-CNTF in PC12 cells.
PC12 cells were stimulated with medium alone, 20 ngÆmL
)1
Hyper-IL-
6, 100 ngÆmL
)1
NGF, 20 ngÆmL
)1

Hyper-CNTF, or 50 ngÆmL
)1
LIF
for 10 min. Cells were lysed in Laemmli buffer and cell lysates were
analyzed for STAT3 and MAPK (p44/p42) phosphorylation as des-
cribed in the legend to Fig. 4. (B) Dose–response of Hyper-CNTF-
induced phosphorylation of STAT3 and MAP kinases in comparison
to CNTF alone, LIF, NGF and Hyper-IL-6. (C) PC12 cells were
activated with 50 ngÆmL
)1
Hyper-CNTF for 10 min up to 4 h. After
lysis, whole cell extracts were Western blotted and their STAT3 and
MAP kinase tyrosine phosphorylation levels were determined.
3028 P. Ma
¨
rz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
pathway. Gp130 activation on different cells can have
multiple physiological consequences such as stimulation of
proliferation, stimulation of differentiation, prevention of
differentiation, prevention of apoptosis and activation of a
family of genes coding for the acute phase proteins [6,49].
The different physiological responses are thought to result
from differential activation of the different intracellular
signal transduction pathways [9,50]. It is not clear to date,
whether these differences are quantitative or temporal. In
other words, signal transduction components might be
overexpressed or underexpressed in different cells. Alter-
natively, the duration of activation of the distinct signal
transduction pathways might be differentially regulated [50].
Interestingly, we have observed that HepG2 cells stimulated

by Hyper-IL-6 showed a more profound and elongated
response as compared to IL-6 [51]. This was most likely due
to decreased internalization of Hyper-IL-6 as compared to
IL-6. We have also recently described differential effects of
IL-6 and Hyper-IL-6 on PC12 cells. Whereas PC12 cells
responded to both IL-6 and Hyper-IL-6 with an increase in
expression of growth associated protein (GAP)-43 mRNA
and protein, only Hyper-IL-6 induced neuronal differenti-
ation in these cells [39]. Intriguingly, it has been shown by
Ihara et al. 1997 [52] that gp130 mutants incapable of
activating the MAPK pathway failed to induce neurite
outgrowth. Consistently, a MAPK kinase inhibitor,
PD98059, inhibited neurite outgrowth. These results suggest
that the activation of the MAPK pathway is essential for
gp130 induced neurite outgrowth of PC12 cells whereas
STAT3 is believed to inhibit this response [52,53]. In line
with these findings, Hyper-CNTF led to a profound
activation of the MAPK pathway with little stimulation
of STAT3. We therefore conclude that upon receptor
stimulation by Hyper-CNTF and LIF in PC12 cells, the
intracellular signal transduction pathways diverge leading to
the observed differences in physiological response in neur-
onal cells. The underlying molecular mechanism might
include the recruitment of the transducing proteins through
binding of Hyper-CNTF and LIF to distinct functional
motifs in the extracellular region of the receptor, leading to
minor conformational changes in the cytoplasmic domains.
Strobl et al. were able to show that for comparable levels of
STAT1 phosphorylation by slightly different chimeric
gp130 receptors, significantly changed transcriptional

responses could be observed indicative for a qualitative
change in the signaling pathway [54].
The newly constructed Hyper-CNTF molecule has two
main advantages over the Hyper-IL-6 and Hyper-IL-11
constructs. First, the spectrum of target cells is more
restricted. All cells in the body express gp130, whereas only
some cells including most cells of the nervous system express
the LIF-R [14]. Therefore, Hyper-CNTF seems to be more
suited for an in vivo application than Hyper-IL-6. Secondly,
the fusion protein Hyper-CNTF does not contain a
synthetic polypeptide linker, the CNTF-R and CNTF being
linked via the flexible C-terminal portion of the CNTF-R
and the N-terminal part of CNTF [33,34] with only a single
additional amino-acid residue introduced. We speculate
that this protein will not be recognized by the immune
system as a foreign protein and should not lead to major
immune responses.
IL-6 type cytokines have been shown to possess robust
neurotrophic activity [14,55–60]. Our data indicate that
Hyper-CNTF in addition to its superagonistic activity,
possesses a unique property over LIF. Therefore, the
possible heightened therapeutic potential of Hyper-CNTF
will have to be tested for nerve regeneration after axotomy
and long-term survival of spinal motoneurons in animal
models.
Administration of CNTF has been shown in various
models with neuromuscular dysfunction to elicit neuropro-
tective effects. For example, CNTF can rescue many motor
neurons in progressive motor neuronopathy pmn mice, a
spontaneous mutant with motor neuronopathy. Moreover,

CNTF has been demonstrated to slow the progression of
motor dysfunction in wobbler mice, another animal model
for motor neuron disease [61]. These findings encouraged
the use of CNTF and related neuropoietic cytokines in
human motor disease. The interest in the neuroprotective
potential of gp130/LIF-R stimulation has been revived by
the demonstration that the CNTF-R not only complexes
with CNTF but also with the newly identified cytokine CLC
[15].
Recently it has been shown that delivery using CNTF-
releasing implants, as described by Aebischer et al. [62–64],
was efficient to treat motor neuron disease in animals. We
propose that similar implants containing recombinant
Hyper-CNTF protein could represent a more optimal way
to stimulate degenerating neuronal cells in amyotrophic
lateral sclerosis or other neurological diseases.
ACKNOWLEDGEMENTS
We thank Dr Birgit Oppmann, Dr Marc Ehlers and Dr Barbara Krebs
for the production of recombinant LIF and CNTF, Dr Thomas
Jostock for cloning of the LIF-R-Fc fusion construct and Dr Hughes
Gascan for the CNTF-R antibody. This work was supported by grants
from the Deutsche Forschungsgemeinschaft (Bonn, Germany), the
Stiftung Rheinland Pfalz fu
¨
r Innovation (Mainz, Germany) and the
Naturwissenschaftlich-Medizinisches Forschungszentrum (Mainz,
Germany) to S. R J., and from the Swiss National Foundation for
Scientific Research (Grant 3100-061571.00/1) and the Deutsche
Forschungsgemeinschaft (SFB505/B5) to U. O.
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