Signaling of Human Ciliary Neurotrophic Factor (CNTF) Revisited
2003; Elsevier BV; Volume: 278; Issue: 11 Linguagem: Inglês
10.1074/jbc.m210044200
ISSN1083-351X
AutoresBjörn Schuster, Marina Kovaleva, Yi Eve Sun, Petra Regenhard, Vance B. Matthews, Joachim Grötzinger, Stefan Rose‐John, Karl‐Josef Kallen,
Tópico(s)Nuclear Receptors and Signaling
ResumoHuman 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 α-receptor (CNTFR), which is not involved in signal transduction, and the β-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 α-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 α-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. 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 α-receptor (CNTFR), which is not involved in signal transduction, and the β-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 α-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 α-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. Ciliary neurotrophic factor (CNTF) 1The 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 was identified as a survival factor for chick ciliary neurons (1Stockli K.A. Lottspeich F. Sendtner M. Masiakowski P. Carroll P. Gotz R. Lindholm D. Thoenen H. Nature. 1989; 342: 920-923Google Scholar, 2Lin L.F. Mismer D. Lile J.D. Armes L.G. Butler E.T.D. Vannice J.L. Collins F. 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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 (50Miller R.G. Bryan W.W. Dietz M.A. Munsat T.L. Petajan J.H. Smith S.A. Goodpasture J.C. Neurology. 1996; 47: 1329-1331Google Scholar). 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 (51Miller 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. Stromatt S.C. Ann. Neurol. 1996; 39: 256-260Google Scholar). 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 (52Schooltink H. Stoyan T. Roeb E. Heinrich P.C. Rose-John S. FEBS Lett. 1992; 314: 280-284Google Scholar, 53Baumann H. Ziegler S.F. 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Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Google Scholar), 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 α-receptor. However, the human IL-6R can serve as a substitute α-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. 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 (55Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Google Scholar). The murine monoclonal antibody (mAb) ab6276 (anti-β-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 (56Sato K. Tsuchiya M. Saldanha J. Koishihara Y. Ohsugi Y. Kishimoto T. Bendig M.M. Cancer Res. 1993; 53: 851-856Google Scholar). Soluble IL-6R and CNTFR were obtained from R&D Systems (Wiesbaden, Germany). [3H]thymidine was from AmershamBiosciences. 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 colibacteria as described before (55Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Google Scholar, 57Jostock T. Blinn G. Renné C. Kallen K.-J. Rose-John S. Müllberg J. J. Immunol. Methods. 1998; 273: 173-181Google Scholar, 58Aasland D. Oppmann B. Grötzinger J. Rose-John S. Kallen K.-J. J. Mol. Biol. 2002; 315: 637-646Google Scholar). 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. 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 mmphenylmethylsulfonyl 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 β-actin. 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 (73Chen G.C. Yang J.T. Anal. Lett. 1977; 10: 1195-1207Google Scholar). 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. 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. The model of the CNTF/IL-6R complex was built using the x-ray structure of CNTF (Ref. 59McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Google Scholar, PDB accession code:1cnt) and the model of the IL-6/IL-6R complex (60Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose John S. Proteins. 1997; 27: 96-109Google Scholar) as a template. In a first step the CNTF molecule was fitted onto the IL-6 model (using only the Cα 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 (61van Gunsteren W.F. Billeter S.R. Eising A.A. Hünenberger P.H. Krüger P. Mark A.E. Scott W.R.P. Tironi I.G. Biomolecular Simulation: The GROMOS96 Manual and User Guide. vdf Hochschulverlag AG, Zürich1996Google Scholar) 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 (62Vriend G. J. Mol. Graph. 1990; 8: 52-56Google Scholar). For graphical representation the Ribbons program was used (63Carson M. J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar). All programs were run on a Silicon Graphics Indy workstation. 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 (55Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Google Scholar). Human CNTF-stimulated proliferation of BaF/3-cells stably transfected with gp130, LIFR, and CNTFR (Fig.1 A), 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. 1 B), 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.1 C). Unexpectedly, however, CNTF showed activity on BaF/3-[gp130,LIFR,IL-6R] cells that expressed the human IL-6R instead of the CNTFR (Fig. 1 D). 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 (55Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Google Scholar), 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. 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 α-receptor (56Sato K. Tsuchiya M. Saldanha J. Koishihara Y. Ohsugi Y. Kishimoto T. Bendig M.M. Cancer Res. 1993; 53: 851-856Google Scholar). PM-1 did not inhibit LIF-induced proliferation of BaF/3-[gp130,LIFR,IL-6R] cells (Fig.2 A), 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. 2 D), 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.Figure 3The anti-IL-6R antibody PM-1 inhibits STAT3 phosphorylation after CNTF stimulation of BaF/3-[gp130/LIFR/IL-6R] cells. STAT3 phosphorylation was measured in BaF/3-[gp130/LIFR/IL-6R]-cells after stimulation with LIF, IL-6, IC7 (all 50 ng/ml), and different concentrations of CNTF in the absence (A) or presence (B) of PM-1 (10 μg/ml). Loading was controlled with an antibody to β-actin.View Large Image Figure ViewerDownload (PPT) Because we and others (53Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Google Scholar, 55Kallen K.-J. Grötzinger J. Lelièvre E. Vollmer P. Aasland D. Renné C. Müllberg J. Meyer zum Büschenfelde K.-H. Gascan H. Rose-John S. J. Biol. Chem. 1999; 274: 11859-11867Google Scholar) have shown, that CNTF is biologically active on HepG2 cells at high concentrations, we wanted to ascertain whether this effect also depended on binding of CNTF to the IL-6R. We therefore stimulated HepG2 cells with increasing amounts of IL-6 and CNTF as well as OSM, which, like LIF, does not require binding to an α-receptor to induce phosphorylation of STAT3 via a gp130/LIFR or gp130/OSMR heterodimer. Similar to our results with BaF/3-[gp130,LIFR,IL-6R] cells, PM-1 competitively inhibited phosphorylation of STAT3 after stimulation with IL-6 and CNTF, but had no effect on the cellular response to OSM (Fig.4, A and B). 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.5 A). BaF/3-[gp130,LIFR] cells were therefore stimulated with CNTF at conc
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