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.     

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 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.     

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.     

Humanin and the Receptors for Humanin

Alzheimer’s disease (AD) is a prevalent dementia-causing neurodegenerative disease. Neuronal death is closely linked to the progression of AD-associated dementia. Accumulating evidence has established that a 24-amino-acid bioactive peptide, Humanin, protects neurons from AD-related neuronal death. A series of studies using various murine AD models including familial AD gene-expressing transgenic mice have shown that Humanin is effective against AD-related neuronal dysfunction in vivo. Most recently, it has been shown that Humanin inhibits neuronal cell death and dysfunction by binding to a novel IL-6-receptor-related receptor(s) on the cell surface involving CNTFRα, WSX-1, and gp130. These findings suggest that endogenous Humanin [or a Humanin-like substance(s)] may suppress the onset of AD-related dementia by inhibiting both AD-related neuronal cell death and dysfunction.     

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.     

No inhibition of IL-27 signaling by soluble gp130

Soluble gp130 is the natural inhibitor of trans-signaling mediated by the soluble IL-6/IL-6R complex. In mouse models, recombinant sgp130 has been successfully applied for the treatment of diseases that are triggered and maintained by soluble IL-6R like Crohn's disease, peritonitis, rheumatoid arthritis, and colon cancer. The novel heterodimeric cytokine IL-27 (p28/EBV-induced gene 3) has been shown to act via a heterodimeric receptor complex of gp130 and the WSX-1 receptor, and to co-regulate the Th(1) immune response after infection. Therefore, we have tested the potential of the recombinant sgp130-Fc protein to also inhibit signaling-mediated IL-27. Here we show that sgp130-Fc does not interfere with IL-27 signaling. We therefore conclude that IL-27 does not bind with high affinity to gp130.     

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.     

Characterization of two putative cytokine receptors, gp130 and ciliary neurotrophic factor receptor, from terrestrial salamanders

Cytokines of the gp130 family are fundamental regulators of immune responses and signal through multimeric receptors to initiate intracellular second-messenger cascades. Here, we provide the first characterization of two full-length gp130 cytokine receptors from the cDNA of the red-legged salamander (Plethodon shermani). The first, gp130 (2745 bp), is a common signaling receptor for several multi-functional cytokines in vertebrates. We also isolated the full-length (1104 bp) sequence of the ciliary neurotrophic factor receptor (CNTFR), which forms a heteromeric signaling complex with gp130. The open reading frames of both receptors were predicted to contain many of the conserved features found in mammalian gp130s, such as cytokine binding homology regions and residues known to form disulfide bonds. Finally, we used RT-PCR to show that gp130 and CNTFR were expressed in most P. shermani tissues, including brain, intestine and muscle. The expression profiles, along with the structural predictions, show that gp130, CNTFR, and their cytokine ligands are parts of the immune system of P. shermani and other caudate amphibians.     

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.     

Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor

Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer''s disease (AD). To mediate the protective effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC₅₀=1–10 μℳ); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC₅₀ of 10–100 pℳ. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ∼5 nℳ. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist.     

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)     

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.     

WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27

The recently discovered cytokine IL-27 belongs to the IL-6/IL-12 family of cytokines and induced proliferation of naive CD4(+) T cells and the generation of a Th1-type adaptive immune response. Although binding of IL-27 to the cytokine receptor WSX-1 was demonstrated, this interaction proved insufficient to mediate cellular effects. Hence, IL-27 was believed to form a heteromeric signaling receptor complex with WSX-1 and another, yet to be identified, cytokine receptor subunit. In this study, we describe that WSX-1 together with gp130 constitutes a functional signal-transducing receptor for IL-27. We show that neither of the two subunits itself is sufficient to mediate IL-27-induced signal transduction, but that the combination of both is required for this event. Expression analysis of WSX-1 and gp130 by quantitative PCR suggests that IL-27 might have a variety of cellular targets besides naive CD4(+) T cells: we demonstrate gene induction of a subset of inflammatory cytokines in primary human mast cells and monocytes in response to IL-27 stimulation. Thus, IL-27 not only contributes to the development of an adaptive immune response through its action on CD4(+) T cells, it also directly acts on cells of the innate immune system.     

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)     

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.     

Differential response of neuronal cells to a fusion protein of ciliary neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory factor.

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 alpha 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 therapeutic potential of Hyper-CNTF as a superagonistic neurotrophin is discussed.