Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor Catherine Guillet1, Eric Lelie`vre1, He´le`ne Plun-Favreau1, Josy Froger1, Marie Chabbert1, J
Trang 1Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor
Catherine Guillet1, Eric Lelie`vre1, He´le`ne Plun-Favreau1, Josy Froger1, Marie Chabbert1, Jacques Hermann1, Amelie Benoit de Coignac2, Jean-Yves Bonnefoy2, Hugues Gascan1, Jean-Franc¸ois Gauchat2
and Greg Elson2,*
1
INSERM U564, CHU d’Angers, Angers, France;2Centre dı´Immunologie Pierre Fabre, St-Julien-en-Genevois, France
The heterodimeric cytokine composed of the soluble ciliary
neurotrophic factor receptor (sCNTFR) and the IL-6
family member cardiotrophin-like cytokine (CLC) was
recently identified as a new ligand for gp130–leukemia
inhibitory factor receptor (LIFR) complex [Plun-Favreau,
H., Elson, G., Chabbert, M., Froger, J., deLapeyriere, O.,
Lelievre, E., Guillet, C., Hermann, J., Gauchat, J F.,
Gascan, H & Chevalier, S (2001) EMBO J 20, 1692–
1703] This heterodimer shows overlapping biological
properties with LIF Although CLC contains a putative
signal peptide and therefore should enter into the classical
secretory pathway, the protein has been shown to be
retained within transfected mammalian cells, unless
coex-pressed with either sCNTFR or cytokine like factor (CLF)
[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 & Gascan,
H (2000) Nat Neurosci 3, 867–872] In the present study,
we demonstrate that a fusion protein comprising CLC covalently coupled through a glycine/serine linker to sCNTFR (CC–FP) is efficiently secreted from transfected mammalian cells CC–FP shows enhanced activities in respect to the CLC/sCNTFR native complex, on a number
of cells expressing gp130 and LIFR on their surface In addition, CC–FP is able to compete with CNTF for cell binding, indicating that both cytokines share binding epitope(s) expressed by their receptor complex Analysis of the downstream signaling events revealed the recruitment
by CC–FP of the signal transducer and activator of tran-scription (STAT)-3, Akt and mitogen-activated protein (MAP) kinase pathways The monomeric bioactive CLC/ sCNTFR fusion protein is therefore a powerful tool to study the biological role of the recently described cytokine CLC
Keywords: CLC; sCNTFR; fusion protein
Ciliary neurotrophic factor (CNTF) was named based on its
ability to maintain the survival of parasympathetic neurons
of chick ciliary ganglions [1,2] Subsequent studies have
revealed that CNTF also enhances the survival of sensory
[3], motor [4], cerebellar and hippocampal neurons [5,6] It
can also prevent lesion-induced degeneration of motor
neurons and slows disease progression in mice with
inherited neuromuscular deficits [7–9] CNTF is also known
to be a trophic factor for skeletal muscles [10,11]
CNTF belongs to a family of structurally related
cytokines that includes leukemia inhibitory factor (LIF),
interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M
(OSM), 1 (CT-1) [12–14] and cardiotrophin-like cytokine (CLC) [15,16] These cytokines share one or both of the receptor signal transducing subunits gp130 or LIF receptor (LIFR) in their respective receptor complexes [17–20] The functional CNTF receptor is a ternary complex, that in addition to gp130 and LIFR also includes
a specificity-determining binding component called CNTF receptor (CNTFR) anchored to the membrane through a glycosylphosphatidylinositol motif [21–25] Binding of the cytokine to the membrane-bound, nonsignaling a chain (CNTFR; [21]), leads to the recruitment of the shared signaling subunits gp130 and the LIFR with the formation
of the high-affinity functional receptor complex [22,23] The subsequent signaling cascade implicates activation of the Janus kinase 1 (JAK1)/STAT3 pathway [26–30]
We recently identified a second ligand for the tripartite CNTF receptor as a complex formed between the IL-6 family cytokine CLC (also known as novel neurotrophin-1/B cell stimulatory factor-3) [15,16], and the soluble type-I cytokine receptor CLF [31,32] We initially observed that CLC, although containing a signal peptide, was inefficiently secreted when expressed in mammalian cells This secretion could be induced upon coexpression with CLF, with the two proteins forming a heterodimer (CLF/CLC) This was the first demonstration of such a secretion mechanism for a cytokine of the IL-6 family and shares certain similarities with the formation of the functional IL-12 heterodimer [33] Like CNTF, CLF/CLC recruits cells
Correspondence to H Gascan: INSERM U564, CHU d’Angers,
4 rue Larrey, 49033 Angers, France.
Fax: + 33 241 73 16 30, Tel.: + 33 241 35 47 29,
E-mail: hugues.gascan@univ-angers.fr or
J.-F Gauchat Centre dı´Immunologie Pierre Fabre,
5 Avenue Napole´on III, 74164 St-Julien-en-Genevois, France.
Fax: + 33 450 35 35 90, Tel.: + 33 450 35 35 55,
E-mail: jean.francois.gauchat@pierre-fabre.com.
Abbreviations: sCNTFR, soluble ciliary neurotrophic factor receptor;
CLC, cardiotrophin-like cytokine; LIFR, leukemia inhibitory factor
receptor; CLF, cytokine like factor; IL, interleukin; MCS, multiple
cloning site; PVDF, poly(vinylidene difluoride); JAK, janus kinase.
*Present address: NovImmune, Geneva, Switzerland.
(Received 8 November 2001, accepted 20 February 2002)
Trang 2expressing on their surface the tripartite CNTF receptor,
induces the tyrosine phosphorylation of gp130, LIFR and
STAT3 in neuroblastoma cells and acts as a survival factor
for motor neurons cultured in vitro [32,34]
We subsequently observed that CLC could also form a
secreted composite cytokine when associated with
sCNTFR Similarly to LIF, CLC/sCNTFR displays
activ-ities on cells which are negative for the expression of
surface-bound CNTFR, but expressing gp130 and LIFR [35]
The association of CLC with sCNTFR is similar to
the situation reported previously for CNTF/sCNTFR,
IL-6/sIL-6R and IL-11/sIL-11R, where composite
cyto-kines implicating a soluble receptor alpha component in
their structure display functional activities mediated
through the appropriate signaling subunits [23,36,37] A
closely related situation also exists for the IL-12 and IL-23
heterodimeric cytokines, composed of an a-receptor-like
chain (p40), respectively associated to p35 or p19 [33,38]
These studies revealed an interesting degree of binding
promiscuity between the IL-6 and IL-12-type ligands and
their multichain receptor complexes
Previous studies have demonstrated that the addition of a
flexible glycine/serine linker between the two subunits of
such composite cytokines allows the expression of a single
chain fusion protein retaining functional activity [38–41]
For example, a fusion protein between IL-6 and soluble
IL-6R, and named ÔHyper IL-6Õ, was shown to be
functionally active in cells where IL-6 alone had no effect
(i.e lacking the membrane-bound form of IL-6R) [39]
These designer molecules display an increased stability
compared with their respective composite cytokines
pre-sumably because the cytokine and its cognate a receptor
component are covalently associated, and both components
remain bound to the signaling receptor subunits for a longer
period of time
In an attempt to facilitate the functional characterization
of the novel neurotrophic complex CLC/sCNTFR, we have
generated a soluble fusion protein CLC is irreversibly
associated to its cognate receptor, the sCNTFR subunit, via
a 10-amino-acid glycine/serine linker We demonstrate that
the CLC/sCNTFR fusion protein is efficiently secreted from
transfected mammalian cells and is highly active on cell
types expressing gp130 and LIFR on their surface
M A T E R I A L S A N D M E T H O D S
Reagents
Human IL-2, CNTF and LIF were purchased from R & D
Systems (Minneapolis, MN, USA) The 4G10 monoclonal
anti-phosphotyrosine Ig was bought from Upstate
Biotech-nology (Lake Placid, NY, USA) and the 9E10 antic-myc
epitope mAb was obtained from the ATCC (Rockville,
MD, USA) The antibody raised against STAT3 was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
Antibodies detecting ERK1/ERK2,
phospho-STAT3 (Tyr705) and phospho-AKT were purchased from
New England Biolabs (Beverly, MA, USA) The
monocl-onal antibodies directed against the human forms of LIFR
(AN-E1, IgG1), gp130 (AN-HH1, IgG2a) and CNTFR
(AN-B2, AN-C2, IgG2a) were generated in the laboratory
[35] The 4–68 monoclonal anti-CNTF Ig was bought from
Roche diagnostics (Meylan, France)
Cell cultures Ba/F3 cells modified to express functional receptors for LIF, CNTF or IL-6 were a kind gift from K J Kallen, University of Kiel, Germany Cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine serum and 5 ngÆmL)1recombinant LIF, CNTF or IL-6 HepG2 hepatoma cells, KB epidermoid carcinoma, HEK 293 cells and SK-N-GP neuroblastoma cells (ATCC, Rockville,
MD, USA) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum
Construction of a single chain CLC/sCNTFR fusion protein (CC–FP)
The cDNA encoding a soluble form of CNTFR (sCNTFR) was amplified by PCR using the primers 5¢-CCGGAATTC GCCAGTGGTGAAGAGATG-3¢ and 5¢-CCGCTCGAG GTCACAGATCTTCGTGGT-3¢, and cloned into the EcoRI and XhoI restriction sites of pcDNA3 The oligonu-cleotides encoding the (G4S)2 flexible polypeptide 5¢-TCG AAGGCGGAGGCGGGAGCGGCGGGGGCGGAAG CGGAGGCGGGGGAAGCCTCGAGT-3¢ and 5¢-CTA GACTCGAGGCTTCCCCCGCCTCCGCTTCCGCCCC CGCCGCTCCCGCCTCCGCCT-3¢, were annealed and cloned into the XhoI and XbaI restriction sites of the afore mentioned pcDNA3 vector containing the sCNTFR cDNA The cDNA encoding a derative of CLC containing the c-myc epitope was amplified from CLC cDNA using the primers 5¢-CCGCTCGAGCTCAATCGCACAGGGGAC CC-3¢ and 5¢-CCGCTCGAGTCAGAGGTCCTCCTCG GAGA-3¢ and cloned into pcDNA3 containing the mod-ified sCNTFR cDNA
Protein purification and Western blotting HEK 293 cell line was stably transfected with pcDNA3 expression vector encoding CC–FP Cell culture medium containing CC–FP was concentrated approximately 10-fold using Centricon-30 units (Millipore, Bedford, MA, USA) and the fusion protein subsequently purified by affinity chromatography using an anti-(c-myc) Ig affinity matrix Bound protein was eluted with 100 mM Glycine-HCl (pH 2.75) A neutral pH was immediately restored using
1MTris base Protein concentrations were determined by SDS/PAGE and silver staining using a BSA protein standard Western-blotting of CC–FP was performed after SDS/PAGE and transfer onto a nylon membrane using a peroxidase coupled anti-(c-myc) Ig
Gel filtration Sample containing CC–FP was fractionated on a Superose
12 size exclusion column Fractions were then analysed by Western-blotting as described before Column calibration was performed using standard purified proteins
Protein modeling CC–FP has been modeled from the molecular models of CLC and CNTFR CLC was modeled from residues 7 to
181 by homology with human CNTF (PDB accession number 1CNT) [42] and with mouse LIF (PDB accession
Trang 3number 1LKI) [43], as described previously [35] Residues
1–286 of CNTFR were modeled by homology with gp130
(PDB accession numbers: 1BQU for the cytokine-binding
domain of gp130 and 1I1R for the Ig-like and the CBD
domains of gp130 in the complex with viral IL-6) [44,45] A
flexible loop including the C-terminal part of CNTFR
(residues 287–316), the linker joining the two proteins
(LEGGGGSGGGGSLE) and the N-terminal part of CLC
(residues 1–6) was generated and refined by simulated
annealing Computations were carried out with the
model-ing program MODELERÒ [46], as implemented in INSIGHT
(MSI, San Diego, USA) on a SGI Octane workstation The
quality of the model was checked withPROFILE3D[47]
Tyrosine phosphorylation analysis
After a 24-h serum starvation, cells were stimulated for
10 min in the presence of the indicated cytokine Cells were
lysed in 10 mM Tris/HCl pH 7.6, 5 mM EDTA, 50 mM
NaCl, 30 mMsodium pyrophosphate, 50 mMsodium
flu-oride, 1 mM sodium orthovanadate, proteinase inhibitors
(1 lgÆmL)1 pepstatin, 2 lgÆmL)1 leupeptin, 5 lgÆmL)1
aprotinin, 1 mMphenylmethanesulfonyl fluoride) and 1%
NP40 or Brij 96 depending on the experiments [35] After
pelleting insoluble material and protein standardization, the
supernatants were immunoprecipitated overnight The
complexes were then isolated with beads coupled to protein
A, submitted to SDS/PAGE and transferred onto an
Immobilon membrane (Millipore, Bedford, MA, USA)
The membranes were subsequently incubated with the
relevant primary antibody before being incubated with the
appropriate secondary antibody labeled with peroxidase for
60 min The reaction was visualized on an X-ray film using
ECL reagents (Amersham, Les Ullis, France) according to
the manufacturer’s instructions In some experiments, the
membranes were stripped overnight in 0.1Mglycine-HCl,
pH 2.7, and neutralized in 1M Tris/HCl, pH 7.6, before
reblotting
Cell proliferation assays
BAF GL (gp130, LIFR), BAF GLC (gp130, LIFR,
CNTFR), BAF gp130/IL-6R or TF1 cells were seeded at
5· 103cellsÆwell)1(in 96-well plates) in RPMI 1640 medium
supplemented with 5% fetal bovine serum containing the
indicated amount of recombinant cytokine Following a
72-h incubation period, a [3H]thymidine pulse was
performed for 4 h and the incorporated radioactivity
determined as described previously [48]
KB cell IL-6 production assay
KB cells were plated in 96-well plates at a concentration of
5· 103cells per well in culture medium containing serial
dilutions of recombinant cytokines as indicated After 48 h,
the supernatants were harvested, and their IL-6 content
determined by ELISA as described previously [32]
Gene reporter assay
Transient transfection of KB cells were carried out in
24-well culture plates using the lipid reagent Fugene 6 from
Roche Diagnostics Cells were transfected with 300 ng
SIEM-luciferase reporter gene, as described previously [32] Forty-eight hours after transfection, cells were incubated with IL-2, LIF, CLC/sCNTFR or CC–FP for an additional
18 h Transfected cells were washed with ice cold NaCl/Pi, and 100 lL of lysis buffer was added to the wells (0.1M
KH2PO4, pH 7.8, 0.1% Triton X-100) Extracts were then used directly to measure the luciferase activity by integrating total light emission over 10 s using a Packard Topcount luminometer (Meriden, CT, USA) Luciferase activity was normalized based on protein concentrations
FACS analysis and cytokine displacement BAF GLC and SK-N-GP cells were incubated in the presence of increasing concentrations of putative competitor (CC–FP, IL-11, IL-4) and a fixed amount of CNTF (2 ng in
a 20-lL final volume) After a 2-h incubation period, cells were washed and incubated with the 4–68 monoclonal anti CNTF Ig, or with an IgG1 control antibody, for 30 min After washing, cells were further incubated with a phy-coerythrin-conjugated anti-(mouse IgG) Ig Fluorescence was subsequently analyzed on a FACScan flow cytometer from Becton & Dickinson (Mountain View, CA, USA)
R E S U L T S
The bioactive designer cytokine hyper-IL-6 (H-IL-6) [39] was used as a model to generate a functional CLC/ sCNTFR complex through monocistronic expression of a CLC/sCNTFR fusion protein (hereafter noted as CC–FP) H-IL-6 is composed of a soluble form of the IL-6 receptor (IL-6R) connected to the mature IL-6 protein via a flexible polypeptide consisting of the glycine/serine linker (G4S)2 The first 16 N-terminal amino acids of IL-6 are nonhelical and therefore presumably flexible, thus contributing to the connecting loop As CNTFR share a high level of sequence homology with IL-6R and CLC shares significant structural homology with IL-6 [15,16], we hypothesized that sCNTFR connected to CLC in a similar fashion would also be functional In contrast to the sIL-6R portion of H-IL-6, the N-terminal signal peptide and Ig-like domain of the sCNTFR precursor protein were maintained in the fusion protein with CLC in order to allow for the secretion of the protein in mammalian cells A c-myc epitope tag was introduced at the C-terminus of the fusion protein for detection and purification purposes (Fig 1A)
HEK 293 cells were stably transfected with an expres-sion vector encoding the fuexpres-sion protein CC–FP was purified from the culture supernatant to apparent homo-geneity with an anti-(c-myc) Ig affinity column (Fig 1B, left panel) SDS/PAGE analysis revealed a single band displaying an apparent molecular weight of 85 kDa, which was quantified based on known concentrations of BSA run
on neighboring lanes Western blotting was employed in order to formally demonstrate that the detected single chain fusion protein effectively was the observed protein (Fig 1B, right panel)
To further check the integrity and folding of the protein, CC–FP was immunoprecipitated with several monoclonal anti-CNTFR Ig, submitted to SDS/PAGE and Western blotted with an anti-(c-myc) Ig As presented in Fig 2A, the mAbs used were able to recognize the protein, indicating that the purified CC–FP was correctly folded
Trang 4To determine whether the protein could self associate to
generate dimers, CC–FP was submitted to a gel filtration
size exclusion column Fractions were then studied by
western-blotting using an anti-(c-myc) Ig The large
major-ity of CC–FP appeared in fraction 17 corresponding to a
molecular mass of 60–75 kDa (Fig 2B) This result
indi-cates that CC–FP preferentially stays as a monomer in
solution
We then tried to determine a molecular model of CC–FP
according to the model of both proteins The
immunoglob-ulin-like domain and the two cytokine binding domains of
CNTFR are represented at the N-terminal side, followed by
a loop containing the glycine linker and, at the C-terminal
end, the four helices of CLC (Fig 2C) In this model, the
putative sites of interaction on CNTFR and CLC are
highlighted in green and red, respectively
The functional properties of CC–FP were tested in
proliferation assays using derivatives of the IL-3-dependent
Ba/F3 cell line, rendered responsive to cytokines of the IL-6
family by transfection of cDNAs encoding the appropriate
receptor chains [48] The CC–FP complex induced a robust
proliferation of Ba/F3 cells expressing LIFR and gp130
(BAF GL), whereas the response observed in the presence
of the CLC/sCNTFR composite cytokine was 10-fold
weaker (Fig 3A) The specific activities were
5· 106UÆmg)1 and 5· 105UÆmg)1, respectively Higher specific activities were observed when using the BAF GLC cells as test cell line (Fig 3B) This is likely to reflect the fact that membrane bound CNTFR is more potent in mediating CLC signaling than its soluble counterpart [34]
Similar experiments were carried out on Ba/F3 cells expressing only gp130, or gp130 and IL-6R on their surface, with no detectable signal being measured in response to either the CLC/sCNTFR composite cytokine, or CC–FP (Fig 3C, and data not shown) This demonstrates an absolute requirement for the LIFR subunit to mediate the signaling response to CLC/sCNTFR or to CC–FP The involvement of gp130 in CC–FP signaling was further confirmed by the inhibition of the BAF GLC proliferative response following the addition to the culture of a neutral-izing monoclonal anti-gp130 Ig (Fig 3D)
We then tested the fusion protein in a second functional assay using the TF1 cell line This is a human erythroleu-kemia cell line known to coexpress LIFR and gp130 on its membrane, but not CNTFR [23] The experiments per-formed revealed a proliferative response to CC–FP similar
to that observed with LIF (Fig 4A) Surprisingly, and in a reproducible manner, we failed to detect any functional effect of the CLC/sCNTFR composite cytokine on TF1 cells This was interpreted as a weaker sensitivity of the gp130–LIFR pathway in the TF1 cell line compared to other LIF-sensitive cell lines In agreement with this observation, the LIF specific activity displayed in TF1 assay was 1.5· 106UÆmg)1whereas it is known to reach
4· 107UÆmg)1using mouse DA1.a cells [49] The CC–FP fusion protein displayed a specific activity of 1· 106UÆmg)1
in the TF1 erythroleukemia assay
The KB carcinoma cell line, which expresses gp130 and LIFR, has been well characterized for its ability to produce IL-6 in response to cytokines signaling via gp130 and LIFR, such as LIF or OSM [32,50] We therefore used KB cells to further characterize the functional activity of CC–FP In accordance with the results obtained in experiments using transfected Ba/F3 cells and TF1 cells, we found CC–FP to
be more potent than CLC/sCNTFR in inducing IL-6 production in KB cells (Fig 4B)
Cytokines signaling through gp130/LIFR can usually compete, at least to some extent, for binding to the same receptor complex [51,52] We examined whether CC–FP and CNTF could be mutually displaced from the cell membrane CNTF binding to BAF GLC and SK-N-GP neuroblastoma cells was monitored by flow cytometry using
a mAb recognizing CNTF (Fig 5A,B) Increasing concen-trations of CC–FP were added with a fixed amount of CNTF A dose-dependent competition for receptor binding was observed using CC–FP, whereas no displacement of CNTF binding was observed with either IL-11 or IL-4 in control experiments These results show that CC–FP and CNTF share binding epitope(s) expressed by their receptor complexes
We next studied the induction of downstream signaling components by the single chain fusion protein following receptor engagement The ability of CC–FP to transduce a signal in cells expressing on their surface the functional LIF receptor complex was subsequently demonstrated The role
of gp130 and LIFR as signaling components for CC–FP was reinforced when analysing their tyrosine phosphoryla-tion following activaphosphoryla-tion by CC–FP in HepG2 hepatoma
Fig 1 Generation of secreted CLC/sCNTFR fusion protein (A)
Schematic representation of the CC–FP fusion protein A cDNA
adaptor encoding a glycine/serine linker (GGGGS) 2 was positioned
between cDNA encoding the sCNTFR protein and CLC sp, signal
peptide (B) Detection of recombinant CC–FP purified from HEK 293
transfected cells In the left panel, purified CC–FP was analyzed by
SDS/PAGE and silver staining of the gel using a BSA protein
stan-dard In the right panel the presence of c-myc epitope-tagged proteins
(CLC/sCNTFR and CC–FP) was detected by Western blotting using
the anti-(c-myc) Ig.
Trang 5Fig 2 Biochemical and structural characterization of the CLC/sCNTFR fusion protein (A) Purified CC–FP was immunoprecipitated with anti-CNTFR Ig (AN-B2, AN-C2), anti-(c-myc) Ig or control mAb as indicated, submitted to SDS/PAGE and Western blotting as described in Fig 1 (B) CC–FP was submitted to a Superose 12 size exclusion column Fractions were analysed by Western blotting using an anti-(c-myc) Ig Column calibration was performed using standard purified proteins (C) Ribbon model of CC–FP: the immunoglobulin-like domain and the two cytokine binding domains of CNTFR are linked to the four helices of CLC by a loop containing the glycine linker (cyan) The putative binding sites of CNTFR and CLC are highlighted in green and red, respectively.
Fig 3 The CLC/sCNTFR fusion protein induces the proliferation of transfected Ba/F3 cells BAF GL (A) and BAF GLC (B) cells were cultured in the presence of serial dilutions of indicated recombinant cytokines Proliferation was measured by [3H]thymidine incorporation and experiments were performed in triplicate Error bars represent the SEM (C) BAF gp130/IL-6R cells were cultured in the presence of serial dilutions of recombinant IL-6, CLC/sCNTFR, CC–FP or IL-2, as control (D) Transfected Ba/F3 cells were incubated in triplicate in culture medium alone (marked as 0), or containing 1 ngÆmL)1CNTF or CC–FP The AN-HH1 Ig (black bars) or a control IgG2a Ig (grey bars) was added at a final concentration of 30 lgÆmL)1.
Trang 6and in SK-N-GP neuroblastoma cells In response to
CC–FP, CLC/sCNTFR and LIF, a clear induction of
tyrosine phosphorylation was detected for gp130 and LIFR
(Fig 6)
A similar result was also observed when analyzing the
activation levels of STAT3 in response to CC–FP and
CNTF (Fig 7A) The transcriptional activity of STAT3
was measured in the KB cell line, which can easily be
transfected in a transient manner For this, cells were
transfected with a reporter construct containing two STAT3
consensus binding sites located upstream of a thymidine
kinase minimal promoter [53] Two days after transfection,
cells were serum starved and stimulated for an additional
15-h with 20 ngÆmL)1of the indicated cytokines Whereas
the CLC/sCNTFR composite cytokine very weakly
stimu-lated STAT3 transcriptional activity, a twofold increase was observed with both CC–FP and LIF (Fig 7B) These results indicate that CC–FP recruits STAT3 to a similar extent than LIF, for both signaling and transcriptional activation
of target genes
It has been previously reported that the PI3-kinase/Akt pathway could regulate gp130 signaling [34] PI3-kinase pathway recruitment by CC–FP led to a marked increase in the tyrosine phosphorylation content of AKT (Fig 8) Comparable results were obtained when treating the cells with CNTF or CLC/sCNTFR In addition to the PI3-kinase/AKT and STAT3 activation pathways, LIFR/gp130 signaling is also known to implicate the GRAB2/Sos adaptators and regulate the MAP kinase pathway [54–56] ERK1 and ERK2, involved in the MAP kinase pathway, have been shown to play important roles in mediating the
Fig 4 Proliferation of the TF1 cells and induction of IL-6 production in
KB cells by CC–FP (A) TF1 cells were cultured in the presence of
serial dilutions of purified recombinant human LIF, CLC/sCNTFR,
CC–FP, or IL-2, as a control Proliferation was measured by
[3H]thymidine incorporation and experiments were performed in
triplicate (B) KB cells were exposed to serial dilutions of CLC/
sCNTFR, CC–FP, LIF or IL-2 as control After a 48-h culture period,
the supernatants were analyzed by ELISA for their IL-6 content.
Experiments were performed in triplicate.
Fig 5 CC–FP and CNTF compete for receptor complex binding BAF GLC and SK-N-GP cells were incubated with 2 ng CNTF and increasing concentrations of CC–FP, IL-11 or IL-2 Detection of CNTF binding was monitored by measuring the mean fluorescence by flow cytometry using an anti-CNTF Ig.
Trang 7mitogenic effects of IL-6 family members ERK1 and ERK2 activation was determined by measuring their tyrosine phosphorylation levels Stimulation of the
SK-N-GP neuroblastoma cell line with CC–FP quickly increased basal values (Fig 8) These results demonstrate the involve-ment of the PI3-kinase/AKT and MAP kinase signaling pathways in functional responses to the CC–FP fusion cytokine
D I S C U S S I O N
We have demonstrated that the fusion of CLC to the C-terminus of sCNTFR via a flexible linker leads to the generation of a bioactive fusion protein Whereas CLC is inefficiently secreted when expressed in the absence of CLF
or CNTFR [32,35], CC–FP is efficiently expressed and secreted in mammalian cells
Similar approaches have been successfully used to gener-ate a number of composite cytokines The first described example reported the generation of a protein consisting of IL-3 fused to GM-CSF, which displayed an increased activity when compared to the respective individual
cytokin-es [57] The discovery of the composite nature of IL-12, encompassing a cytokine-like component (p35) associated to
Fig 8 Analysis of AKT and ERK1, ERK2 tyrosine phosphorylation induced by CC–FP SK-N-GP cells were incubated either with or without 50 ngÆmL)1 of CNTF, CLC/sCNTFR and CC–FP for
10 min After lysis in 1% NP-40, lysates were subjected to immunoblot analysis with antibodies specific for activated AKT (AKT-P) or recognizing activated forms of ERK1 and ERK2 (ERK1-P and ERK2-P).
Fig 7 Analysis of STAT3 tyrosine phosphorylation and transcriptional
activation induced by CC–FP (A) CC–FP induces STAT3 tyrosine
phosphorylation in SK-N-GP neuroblastoma and HepG2 cells
Fol-lowing a 10-min exposure to either NaCl/P i (marked as 0), CNTF
(50 ngÆmL)1), CLC/sCNTFR (50 ngÆmL)1), or purified CC–FP
(50 ngÆmL)1), cells were lysed and subject to Western blotting using an
anti-(STAT3-P) mAb (B) Effect of CC–FP stimulation on STAT3
transcriptional activity KB carcinoma cells were transiently
trans-fected with a reporter plasmid gene (SIEM-luciferase) 48 h later, cells
were treated with 20 ngÆmL)1of LIF, CLC/sCNTFR, CC–FP or IL-2
as a control, for an additional 18 h Cellular extracts were prepared
and used to directly measure luciferase activity.
Fig 6 Analysis of gp130 and LIFR tyrosine phosphorylation induced by CC–FP CC–FP induces gp130 and LIFR tyrosine phospho-rylation in SK-N-GP neuroblastoma and in HepG2 cells Following a 10-min exposure to either NaCl/P i (marked as 0), LIF
(50 ngÆmL)1), CLC/sCNTFR (50 ngÆmL)1),
or purified CC–FP (50 ngÆmL)1), cells were lysed and subject to immunoprecipitation (IP) using an anti-LIFR Ig and Western blotting (WB).
Trang 8a soluble receptor-like subunit (p40), opened the possibility
of fusing the two components, or even adding an
immuno-globulin portion to fused IL-12 to reinforce the targeting of
the cytokine towards a defined cell type [41,58,59] Designed
IL-12 fusion proteins do not display any increase in their
specific activity, when compared to the wild type protein
This is in part explained by the fact that p35 and p40
components are already covalently associated through a
disulfide bridge leading to a stable association
Many examples of cytokine receptors existing in soluble
form in vivo have been reported Almost all of these soluble
receptors are able to interfere with the activity of their
ligands An interesting feature of the a receptor components
belonging to the IL-6 family is their ability to promote
the action of their ligands These have been described in
detail for IL-6/IL-6R, CNTF/CNTFR and IL-11/IL-11R
[23,36,37] For this cytokine family the ligand–receptor
interaction is mainly governed by the dissociation rate,
suggesting that the average half-life of the cytokine–soluble
receptor complex may be shorter than the time required to
recruit the larger signaling receptors, gp130, LIFR or
OSMR Accordingly, fused proteins interacting with their
cognate receptor show a lower off-rate leading to a stronger
recruitment of the signaling subunits
As the CC–FP fusion protein represents an irreversibly
bound and therefore stabilized derivative of its respective
complex, it is noteworthy that CC–FP displays enhanced
biological activity relative to CLC/sCNTFR On cells
expressing gp130 and LIFR, the CC–FP fusion protein
was shown to be 10- to 100-fold more potent when compared
to the unlinked composite cytokine This result corroborates
previous studies showing that single-chain fusion proteins
between cytokines and their nonsignaling binding receptors
exhibit enhanced functional activity with respect to their
native cytokine/receptor complexes [39,60–63]
In the present study we analyzed the proliferative
potential of CC–FP in cells displaying a hematopoietic
background (TF1 and Ba/F3 cells) CC–FP also activates
these cells by increasing their transcriptional machinery
leading to specific protein synthesis, such as acute phase
protein synthesis for hepatocytes [64], or IL-6 production in
the case of the KB epidermoid carcinoma [50] Collectively,
these results support the idea that CC–FP should be able to
substitute for LIF in a large number of situations It is worth
underlining the synergistic potential of gp130 activating
cytokines together with SCF, GM-CSF and
erythropoie-tin in increasing the maintenance and proliferation of
CD34+CD38– or CD34+Thy1+ hematopoietic stem
cells in vitro [65,66] Therefore, the involvement of CC–FP
in hematopoietic stem cell expansion is currently under
investigation
CNTF promotes the differentiation and survival of a
wide range of cell types in the nervous system [1–6] We can
therefore assume that composite CLC-containing cytokines
will display overlapping functions Although CLC uses the
same functional receptor as CNTF, it differs from the latter
in that it is apparently naturally secreted under
nontrau-matic conditions [32,35] Recent studies reported the
possibility of expanding human central nervous system
stem cells by in vitro growth [67,68] Developed cultures can
continuously propagate a heterogeneous population of
early neural stem and/or progenitor cells Experiments have
been carried out with neurosphere cultures, requiring a
cocktail of cytokines Among them, LIF was shown to play
an important role for correct culture development The availability of a CLC/sCNTFR fusion protein using the LIF signaling receptor complex should be of great interest in this context
Due to its neuroprotective effects, much investigation has been made into the potential utility of CNTF in the treatment of neurodegenerative disorders such as amyo-trophic lateral sclerosis and Huntington’s disease Indeed, promising preclinical studies have led to clinical trials with varying success [69–72] The high level of toxicity upon systemic injection of the protein has led to the targeted administration of CNTF to the CNS via intrathecal implantation of encapsulated transfected cells For this reason, similar studies should be performed to determine the ability of CLC/sCNTFR to convey neuroprotective effects whilst assessing its toxicity The availability of a monomeric bioactive CC–FP fusion protein should therefore allow the production of sufficient purified protein and facilitate the generation of stable transfected cell lines and vectors for alternative gene therapy approaches
A C K N O W L E D G E M E N T S
He`le`ne Plun-Favreau and Catherine Guillet were funded by grants from the city of Angers and the Departement du Maine et Loire, respectively The project was supported by a grant from the Association Franc¸aise contre les Myopathies.
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