Gene expression of receptors for IL-6, LIF, and CNTF in regenerating skeletal muscles.

The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor alpha (IL-6R), LIF receptor beta (LIFR), and CNTF receptor alpha (CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons. (J Histochem Cytochem 48:1203-1213, 2000)     

Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex

The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor alpha (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex.     

Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF

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 alpha-receptor (CNTFR), which is not involved in signal transduction, and the beta-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 alpha-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 alpha-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.     

Expression of biologically active mouse ciliary neutrophic factor (CNTF) and soluble CNTFRalpha in Escherichia coli and characterization of their functional specificities

Ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine initially identified in chick embryo. It has been evaluated for the treatment of neurodegenerative diseases. CNTF also acts on non-neuronal cells such as oligodendrocytes, astrocytes, adipocytes and skeletal muscles cells. CNTF has regulatory effects on body weight and is currently in clinical trial for the treatment of diabetes and obesity. CNTF mediates its function by activating a tripartite receptor comprising the CNTF receptor alpha chain (CNTFRalpha), the leukemia inhibitory factor receptor beta chain (LIFRbeta) and gp130. Human, rat and chicken CNTF have been expressed as recombinant proteins, and most preclinical studies in murine models have been performed using rat recombinant protein. Rat and human CNTF differ in their fine specificities: in addition to CNTFR, rat CNTF has been shown to activate the LIFR (a heterodimer of LIFRbeta and gp130), whereas human CNTF can bind and activate a tripartite receptor comprising the IL-6 receptor alpha chain (IL-6Ralpha) and LIFR. To generate tools designed for mouse models of human diseases; we cloned and expressed in E. coli both mouse CNTF and the CNTFRalpha chain. Recombinant mouse CNTF was active and showed a high level of specificity for mouse CNTFR. It shares the arginine residue with rat CNTF which prevents binding to IL-6Ralpha. It did not activate the LIFR at all concentrations tested. Recombinant mouse CNTF is therefore specific for CNTFR and as such represents a useful tool with which to study CNTF in mouse models. It appears well suited for the comparative evaluation of CNTF and the two additional recently discovered CNTFR ligands, cardiotrophin-like cytokine\cytokine-like factor-1 and neuropoietin.     

A peptide derived from the CD loop-D helix region of ciliary neurotrophic factor (CNTF) induces neuronal differentiation and survival by binding to the leukemia inhibitory factor (LIF) receptor and common cytokine receptor chain gp130

Ciliary neurotrophic factor (CNTF) induces neuronal differentiation and promotes the survival of various neuronal cell types by binding to a receptor complex formed by CNTF receptor α (CNTFRα), gp130, and the leukemia inhibitory factor (LIF) receptor (LIFR). The CD loop-D helix region of CNTF has been suggested to be important for the cytokine interaction with LIFR. We designed a peptide, termed cintrofin, that encompasses this region. Surface plasmon resonance analysis demonstrated that cintrofin bound to LIFR and gp130, but not to CNTFRα, with apparent KD values of 35 nM and 1.1 nM, respectively. Cintrofin promoted the survival of cerebellar granule neurons (CGNs), in which cell death was induced either by potassium withdrawal or H2O2 treatment. Cintrofin induced neurite outgrowth from CGNs, and this effect was inhibited by specific antibodies against both gp130 and LIFR, indicating that these receptors are involved in the effects of cintrofin. The C-terminal part of the peptide, corresponding to the D helix region of CNTF, was shown to be essential for the neuritogenic action of the peptide. CNTF and LIF induced neurite outgrowth in CGNs plated on laminin-coated slides. On uncoated slides, CNTF and LIF had no neuritogenic effect but were able to inhibit cintrofin-induced neuronal differentiation, indicating that cintrofin and cytokines compete for the same receptors. In addition, cintrofin induced the phosphorylation of STAT3, Akt, and ERK, indicating that it exerts cell signaling properties similar to those induced by CNTF and may be a valuable survival agent with possible therapeutic potential.     

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

Expression of CNTF/LIF‐receptor components and activation of STAT3 signaling in axotomized facial motoneurons: Evidence for a sequential postlesional function of the cytokines

Several lines of evidence suggest that ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are important for the survival and regeneration of axotomized motoneurons. To investigate the role of CNTF/LIF signaling in regenerative responses of motoneurons, we studied the expression of the three receptor components, CNTF receptor α (CNTFRα), LIF receptor β (LIFRβ), and gp130, and the activation of the STAT3 signal transduction pathway in the rat facial nucleus following peripheral nerve transection. As shown by in situ hybridization and immunoblotting, axotomy resulted in a rapid down‐regulation of CNTFRα mRNA expression within 24 h and a concomitant massive up‐regulation of LIFRβ mRNA and protein in the lesioned motoneurons. The altered mRNA levels were maintained for 3 weeks but had returned back to control levels by 6 weeks postlesion after successful regeneration. In contrast, mRNA levels remained in the lesioned state during the 6‐week period studied, when regeneration was prevented by nerve resection. Significant lesion‐induced changes in gp130 mRNA levels were not detectable. Rapid (within 24 h) and sustained (for at least 5 days) activation of STAT3 in axotomized facial motoneurons was revealed by demonstrating the phosphorylation and nuclear translocation of the protein using immunocytochemistry and immunoblotting. In agreement with previous studies showing a complementary regulation of CNTF and LIF in the lesioned facial nerve, our observations on the postlesional regulation of CNTF/LIF receptor components in the facial nucleus indicate a direct and sequential action of the two neurotrophic proteins on axotomized facial motoneurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 559–571, 1999     

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.     

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.     

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin II-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR. (Mol Cell Biochem 269: 95–101, 2005)     

Membrane distal cytokine binding domain of LIFR interacts with soluble CNTFR in vitro

Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of the CNTF receptor alpha (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand-receptor interactions are mediated through the cytokine binding domains (CBDs) and/or the immunoglobulin-like domains of the receptors. However, in the case of CNTF, the precise nature of the protein-protein contacts in the signaling complex has not yet been resolved. In this study, we provide the first demonstration that the membrane distal CBD (CBD1) of LIFR associates in vitro with soluble CNTFR in the absence of CNTF. Moreover, purified CBD1 partially blocks CNTF signaling, but not that of interleukin-6 or LIF, in human embryonal carcinoma cell line Ntera/D1 cells. These data raise the possibility that LIFR has the capability to form a ligand-free complex with CNTFR.     

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.     

CNTF mediates neurotrophic factor secretion and fluid absorption in human retinal pigment epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (J(V)) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease.     

Regulation of neurokine receptor signaling and trafficking

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are neurally active cytokines, or neurokines. LIF signals through a receptor consisting of gp130 and the low affinity LIF receptor (LIFR), while the CNTF receptor consists of gp130, LIFR, and the low affinity CNTF receptor (CNTFR). Ser1044 of the LIFR is phosphorylated by Erk1/2 MAP kinase. Stimulation of neural cells with growth factors which strongly activate Erk1/2 decreases LIF-mediated signal transduction due to increased degradation of the LIFR as a consequence of Erk1/2-dependent phosphorylation of the receptor at Ser1044.     

Humanin Inhibits Neuronal Cell Death by Interacting with a Cytokine Receptor Complex or Complexes Involving CNTF Receptor α/WSX-1/gp130

Humanin (HN) inhibits neuronal death induced by various Alzheimer''s disease (AD)-related insults via an unknown receptor on cell membranes. Our earlier study indicated that the activation of STAT3 was essential for HN-induced neuroprotection, suggesting that the HN receptor may belong to the cytokine receptor family. In this study, a series of loss-of-function tests indicated that gp130, the common subunit of receptors belonging to the IL-6 receptor family, was essential for HN-induced neuroprotection. Overexpression of ciliary neurotrophic factor receptor α (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it. These results suggest that both CNTFR and WSX-1 may be also involved in HN binding to cells. Consistent with these results, loss-of-functions of CNTFR or WSX-1 in neuronal cells nullified their responsiveness to HN-mediated protection. In vitro–reconstituted binding assays showed that HN, but not the other control peptide, induced the hetero-oligomerization of CNTFR, WSX-1, and gp130. Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130.     

Minimal Interleukin 6 (IL-6) Receptor Stalk Composition for IL-6 Receptor Shedding and IL-6 Classic Signaling

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) is coordinated by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The soluble IL-6R is mainly generated by ADAM10- and ADAM17-mediated ectodomain shedding. Little is known about the role of the 52-amino acid-residue-long IL-6R stalk region in shedding and signal transduction. Therefore, we generated and analyzed IL-6R stalk region deletion variants for cleavability and biological activity. Deletion of 10 amino acids of the stalk region surrounding the ADAM17 cleavage site substantially blocked IL-6R proteolysis by ADAM17 but only slightly affected proteolysis by ADAM10. Interestingly, additional deletion of the remaining five juxtamembrane-located amino acids also abrogated ADAM10-mediated IL-6R shedding. Larger deletions within the stalk region, that do not necessarily include the ADAM17 cleavage site, also reduced ADAM10 and ADAM17-mediated IL-6R shedding, questioning the importance of cleavage site recognition. Furthermore, we show that a 22-amino acid-long stalk region is minimally required for IL-6 classic signaling. The gp130 cytokine binding sites are separated from the plasma membrane by ∼96 Å. 22 amino acid residues, however, span maximally 83.6 Å (3.8 Å/amino acid), indicating that the three juxtamembrane fibronectin domains of gp130 are not necessarily elongated but somehow flexed to allow IL-6 classic signaling. Our findings underline a dual role of the IL-6R stalk region in IL-6 signaling. In IL-6 trans-signaling, it regulates proper proteolysis by ADAM10 and ADAM17. In IL-6 classic-signaling, it acts as a spacer to ensure IL-6·IL-6R·gp130 signal complex formation.