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, 101 (14), 4827-32

Neuropoietin, a New IL-6-related Cytokine Signaling Through the Ciliary Neurotrophic Factor Receptor

Affiliations

Neuropoietin, a New IL-6-related Cytokine Signaling Through the Ciliary Neurotrophic Factor Receptor

Damien Derouet et al. Proc Natl Acad Sci U S A.

Abstract

A structural profile-based computational screen was used to identify neuropoietin (NP), a new cytokine. The np gene is localized in tandem with the cardiotrophin-1 gene on mouse chromosome 7. NP shares structural and functional features with ciliary neurotrophic factor (CNTF), cardiotrophin-1, and cardiotrophin-like cytokine. It acts through a membrane receptor complex comprising CNTF receptor-alpha component (CNTFRalpha), gp130, and leukemia inhibitory factor receptor to activate signal transducer and activator of transcription 3 signaling pathway. NP is highly expressed in embryonic neuroepithelia. Strikingly, CNTFRalpha, but not its alternate ligands, CNTF and cardiotrophin-like cytokine, is expressed at the same developmental stages. NP is also observed in retina and to a lesser extent in skeletal muscle. Moreover, NP could sustain the in vitro survival of embryonic motor neurons and could increase the proliferation of neural precursors when associated to epidermal growth factor and fibroblast growth factor 2. Thus, NP is a new ligand for CNTFRalpha, with important implications for murine nervous system development.

Figures

Fig. 5.
Fig. 5.
Tripartite functional receptor for NP. (A) Silver staining and Western blot analyses of NP purified from a Ni+ agarose column. (B) Proliferation of transfected Ba/F3 cells to NP. Murine IL-3 was used as positive control for parental-, LIFR-, and CNTFRα-transfected Ba/F3 cells, whereas IL-6/sol.IL-6R, LIF, and CNTF were used as controls for BAF gp130, Ba/F3 cells expressing only gp130 and LIFR, and BAF GLC, respectively (most of the SE deviation values were inferior to the symbol size). (C) Inhibition of NP-induced BAF GLC cell proliferation by using neutralizing anti-gp130 and LIFR Abs. Cells were incubated with 5 ng/ml NP (black bars) or murine IL-3 as control (open bars), alone (none) or with 15 μg/ml of an anti-gp130 (AN-HH1), anti-LIFR (12D3), or an IgG-matched control mAb, as indicated. (D) NP sustains motor neuron survival. Survival effects were measured on motor neurons from E14.5 rat embryos maintained in culture for 3 days in the presence of indicated cytokine concentrations, in ng/ml. (E) Proliferation of human TF1 and CNTFRα-transfected TF1 cells to NP. Human LIF was used as a positive control for both studied cell lines (most of the SE deviation values were inferior to the symbol size).
Fig. 1.
Fig. 1.
NP amino acid sequence and its comparison with related IL-6 family members. (A) Sequence alignment of mouse NP with mouse CT-1, CNTF, and CLC. Strictly conserved and 80% conserved amino acids are in red and blue, respectively. Black bars indicate the four predicted α-helices. Predicted signal peptide is highlighted with a dashed line. An asterisk represents a potential N-glycosylation site on NP. (B) Alignment of concatened exons of mouse, rat, chimpanzee, and human NP orthologs. Indicative exon positions were based on mouse np sequence. A dashed box indicates the predicted signal peptide-coding regions. A plain box highlights the deletion in the putative third exon of human NP.
Fig. 2.
Fig. 2.
Evolution of the np gene. (A) Evolutionary dendrogram for members of the IL-6 family cytokines. (B) Gene localization of mouse np on chromosome 7F3 located in tandem with ct-1. Positions are expressed in kilobytes. (C) Exon/intron organization of np gene.
Fig. 3.
Fig. 3.
Expression profiles of NP. (A) NP expression was studied by RT-PCR in adult mice tissues and in E15 brain. NP plasmid DNA was used as positive control. (B) Expression of NP, CLC, CNTF, and CNTFRα were measured in developing embryo by quantitative PCR, as described in Materials and Methods. (C) In situ localization of NP mRNA in mouse embryonic tissues. (a) Transverse section of the lateral ventricle of E12.5 embryo. (c) Sagittal section through E16 olfactory neuroepithelium. NP signal was detected throughout neuroepithelia in scattered cells as shown in higher magnifications of squares in a (b) and c (d). (e) Sagittal section through E14.5 retina. (f) Higher magnification of the square in e showing that NP expression is dispersed throughout the retina in both inner nuclear layer and outer nuclear layer where neural retina progenitors are actively differentiating. (g) Transverse section of forelimb muscles from E14.5 MLCnlacZ mice, which express the nlacZ-reporter gene in muscles under the control of myosin light-chain promoter, double stained by in situ hybridization for NP and by immunohistochemical staining for β-galactosidase. β-galactosidase-positive cells (brown nuclear staining) are also weakly NP positive (blue staining). (h and i) Sagittal sections through trigeminal ganglion hybridized for NP (h) or CNTFRα (i). ne, neuroepithelium; lv, lateral ventricle; oe, olfactory epithelium; inl, inner nuclear layer; onl, outer nuclear layer; d, dermis; mu, muscle; tr, trigeminal ganglion. (Scale bars: 100 μm for a, c, e, and gi;30 μm for b, d, and f.)
Fig. 4.
Fig. 4.
Proliferation of neurospheres to NP. (A) Proliferation assay of neurospheres by using serial dilution of CNTF (▵) or NP(▪) in presence of 20 ng/ml EGF and FGF2 (some of the SE deviation values were inferior to the symbol size). In the absence of EGF and FGF2, or by using NP alone, the obtained values were 305 and 2,153 cpm, respectively. (B) Phase-contrast photomicrograph of neurosphere cultures supplemented with 20 ng/ml EGF and FGF2 (Left) or with 20 ng/ml EGF and FGF2 plus 25 ng/ml NP (Right) during 7 days. (Scale bar, 50 μm.)
Fig. 6.
Fig. 6.
Physical interaction and recruitment of CNTF receptor complex by NP. (A) Coimmunoprecipitation of NP and CNTFRα from the surface of BAF GLC cells. BAF GLC were incubated with IL-2 (50 ng/ml)-, CNTF (50 ng/ml)-, mock-, or NP-containing culture media for 10 min at 37°C. Cells were lysed with Brij 96 buffer, and proteins were immunoprecipitated with AN-D3 anti-CNTFRα mAb and protein A beads. NP was detected by using an anti-V5 epitope mAb. (B) Coimmunoprecipitation of CNTF (Upper) or NP(Lower) with CNTFRα-Fc, gp130-Fc, or LIFR-Fc fusion proteins. Receptor subunits were incubated at a concentration of 4 nM for 16hat4°C in the presence of 4 nM NP or CNTF before being immunoprecipitated with protein A beads. NP and CNTF were detected by using an anti-V5 epitope mAb and a polyclonal biotinylated anti-CNTF Ab, respectively. (C) Tyrosine phosphorylation of gp130 and LIFR in SK-N-GP cells in response to NP. After an exposure to 50 ng/ml of indicated cytokines, SK-N-GP cells were lysed with Brij 96 buffer. Then, LIFR and gp130 were coprecipitated by using the AN-E1 anti-LIFR mAb. Tyrosine phosphorylation content was determined with the 4G10 anti-phosphotyrosine mAb. (D) Induction of STAT3 tyrosine phosphorylation by NP in SK-N-GP neuroblastoma (gp130+, LIFR+, and CNTFRα+) and T98G glioblastoma cells (gp130+, LIFR+, and CNTFRα–). After exposure to 50 ng/ml of indicated cytokines, cells were lysed with 1% Nonidet P-40 buffer and analyzed by Western blotting by using an anti-phospho-STAT3 Ab.

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