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Human ciliary neurotrophic factor: a structure-function analysis.

ABSTRACT: Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons. The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices. CNTF exerts its activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction [gp130 and leukaemia inhibitory factor receptor (LIF-R)]. We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein. In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity. In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.

SUBMITTER: Kruttgen A

PROVIDER: S-EPMC1135822 | biostudies-other | 1995 Jul

REPOSITORIES: biostudies-other

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Project description:Administration of ciliary neurotrophic factor (CNTF) to experimental animals exerts anti-obesity effects by acting on multiple targets. In white adipose tissue CNTF reduces lipid content, promotes fatty acid (FA) oxidation and improves insulin sensitivity. This study was performed to establish whether CNTF exerts similar effects on human white adipocytes. To this end, adipose differentiation was induced in vitro in human multipotent adipose-derived stem (hMADS) cells. CNTF receptor α (CNTFRα) expression was assessed in hMADS cells and adipocytes by qRT-PCR, Western blotting, and immunocytochemistry. After administration of human recombinant CNTF, signaling pathways and gene expression were evaluated by Western blotting and qRT-PCR. Glucose uptake was assessed by measuring 2-nitrobenzodeoxyglucose uptake with a fluorescence plate reader. Lastly, CNTF-induced anti-inflammatory responses were evaluated in hMADS adipocytes stressed with tumor necrosis factor α (TNFα) for 24 h. Results showed that CNTFRα protein expression was higher in undifferentiated hMADS cells than in hMADS adipocytes, where it was however clearly detectable. In hMADS adipocytes, 1 nM CNTF strongly activated the JAK-STAT3 (Janus kinase-signaling transducer and activator of transcription 3) pathway and acutely and transiently activated the AMPK (AMP-activated protein kinase) and AKT (protein kinase B) pathways. Acute CNTF treatment for 20 min significantly increased basal glucose uptake and was associated with increased AKT phosphorylation. Longer-term (24 and 48 h) treatment reduced the expression of lipogenic markers (FA synthase and sterol regulatory element-binding protein-1) and increased the expression of lipolytic [hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL)] and mitochondrial (peroxisome proliferator-activated receptor γ coactivator-1α and carnitine palmitoyltransferase 1) markers. In TNFα-treated hMADS adipocytes, CNTF significantly reduced the expression of monocyte chemoattractant protein 1 and TNFα-induced AKT inhibition. Collectively, these findings demonstrate for the first time that CNTF plays a role also in human adipocytes, driving their metabolism toward a less lipid-storing and more energy-consuming phenotype.

Project description:Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans through activation of the hypothalamic Jak-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway. Here, we tested the hypothesis that CNTF also affects the brainstem centers involved in feeding and energy balance regulation. To this end, wild-type and leptin-deficient (ob/ob and db/db) obese mice were acutely treated with intraperitoneal recombinant CNTF. Coronal brainstem sections were processed for immunohistochemical detection of STAT3, STAT1, STAT5 phosphorylation and c-Fos. In wild-type mice, CNTF treatment for 45 min induced STAT3, STAT1, and STAT5 phosphorylation in neurons as well as glial cells of the area postrema; here, the majority of CNTF-responsive cells activated multiple STAT isoforms, and a significant proportion of CNTF-responsive glial cells bore the immaturity and plasticity markers nestin and vimentin. After 120 min CNTF treatment, c-Fos expression was intense in glial cells and weak in neurons of the area postrema, it was intense in several neurons of the rostral and caudal solitary tract nucleus (NTS), and weak in some cholinergic neurons of the dorsal motor nucleus of the vagus. In the ob/ob and db/db mice, Jak-STAT activation and c-Fos expression were similar to those induced in wild-type mouse brainstem. Treatment with CNTF (120 min, to induce c-Fos expression) and leptin (25 min, to induce STAT3 phosphorylation) demonstrated the co-localization of the two transcription factors in a small neuron population in the caudal NTS portion. Finally, weak immunohistochemical CNTF staining, detected in funiculus separans, and meningeal glial cells, matched the modest amount of CNTF found by RT-qPCR in micropunched area postrema tissue, which in contrast exhibited a very high amount of CNTF receptor. Collectively, the present findings show that the area postrema and the NTS exhibit high, distinctive responsiveness to circulating exogenous and, probably, endogenous CNTF.

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Human ciliary neurotrophic factor: a structure-function analysis.

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