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: p.maerz@unibas.ch 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. 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