Differentiation Effects of Ciliary Neurotrophic Factor on Human Neuroblastoma Cells

Abstract: In the human neuroblastoma cell line LA-N-2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half-maximally saturated at <100 p M of the neurokine, but was not required for cell survival in serum-free conditions over a 13-day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high-affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross-linking studies were consistent with tripartite receptor activation as a mediator of the observed biological effects.     

Nerve Growth Factor Receptors in Human Neuroblastoma Cells

Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN-1 were characterized. LAN-1 cells display high-affinity (type I, with KD value of 5.9 X 10(-11) M) and low-affinity (type II, with KD value of 9.2 X 10(-9) M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High-affinity binding was found in the 5.9-6.2 pH region of the PEGG, and low-affinity binding in the 4.6-4.8 and 8.8-9.3 pH ranges. After further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we observed both 92.5- and 200-kDa molecular species associated with NGF binding activity. The 200-kDa protein was found in fractions displaying high-affinity NGF binding and the 92.5-kDa protein in fractions displaying low-affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high-affinity NGF binding and a decrease in low-affinity NGF binding. Immunoprecipitation of this "mixed" fraction and analysis on SDS-PAGE under reduced and nonreduced conditions showed 200-kDa and 92.5-kDa proteins under nonreduced conditions and a 92.5-kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF-responsive cells. The interconvertibility of low- and high-affinity receptors and the possible existence of a modulator type protein or of "silent" type receptors are also in agreement with our findings.     

Differential Effects of Nerve Growth Factor and Ciliary Neuronotrophic Factor on Catecholamine Storage and Catecholamine Synthesizing Enzymes of Cultured Rat Chromaffin Cells

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.     

Effects of a Peptide Analogue of the Amphiphilic Domain of the Common Neurotrophin Receptor on Nerve Growth Factor-Mediated Motility of Human Neuroblastoma Cells

Abstract: Exposure of human neuroblastoma cells (IMR-32) to a peptide mimic of the cytoplasmic amphiphilic domain of the common neurotrophin receptor (p75^NTR 367–379) resulted in enhanced nerve growth factor (NGF)-mediated inhibition of cell invasion in vitro. The peptide also enhanced NGF-mediated neurite extension and GAP-43 gene expression but had no effect on NGF-mediated cell survival. These latter functional effects mimicked influences on NGF-mediated neurite growth in other trkA-positive cells as reported previously. NGF-dependent trkA phosphorylation was significantly enhanced by the presence of the peptide, whereas high-affinity binding of ¹²⁵I-NGF, both NGF receptors mRNA and protein expression, and trkA dimer/monomer ratios were not influenced. The studies suggest that ligand-mediated trkA activation has differential effects on cell motility phenomena and that the amphiphilic domain of p75^NTR has a role in this differential signaling.     

人睫状神经营养因子基因及突变体在大肠杆菌中的表达

人睫状神经营养因子（ｈｕｍａｎ ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ｈＣＮＴＦ）ｃＤＮＡ克隆入具有ＰＬ启动子的表达载体,转化大肠杆菌ＨＢ１０１,构建成表达ｈＣＮＴＦ菌株。热诱导后,ｈＣＮＴＦ的表达量达菌体总蛋白量的２５％。用离子交换层析、凝胶过滤层析从菌体裂解液中纯化了ｈＣＮＴＦ。ＳＤＳ－ＰＡＧＥｈＣＮＴＦ的分子量约为２４ｋｄ,Ｎ端氨基酸序列分析结果与依基因核苷酸推导疗列相符。     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Defining Responsiveness of Avian Cochlear Neurons to Brain-Derived Neurotrophic Factor and Nerve Growth Factor by HSV-1-Mediated Gene Transfer

Abstract: The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV-1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV-1-mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF-responsive by transducing them with the high-affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.     

Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme-Linked Immunoassay

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.     

Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.     

Brain-Derived Neurotrophic Factor Is More Highly Conserved in Structure and Function than Nerve Growth Factor During Vertebrate Evolution

Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fish Xiphophorus, with the mammalian homologues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purified recombinant BDNF. However, the activity of recombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng/ml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF had reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates from a common ancestral gene about 600 million years ago. By RNA gel blot analysis NGF mRNA was detected during late embryonic development; BDNF was present in adult brain.     

Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor Increase Activated Ras in a Neuroblastoma Cell Line and in Sympathetic Neuron Cultures

Abstract: The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.     

重组人睫状神经营养因子的聚乙二醇化修饰简

目的：研究重组人睫状神经营养因子（rhCNTF）突变体的聚乙二醇（PEG）化修饰,对rhCNTF的PEG化产物进行初步分离纯化及相关生物活性检测。方法：采用分子生物学技术经点突变得到rhCNTF的突变体cNm通过实验设计研究CN10的最佳PEG化条件;采用分子筛层析方式对偶联产物进行初步纯化,最后用ELISA和小鼠体重增长抑制法检测PEG化后的CN。。蛋白的生物活性。结果：能运用mPEG—MAL对CN,。进行定点修饰,PEG化后用Superdex200能够分离CN10;PEG化后的CN10每2d腹腔注射1次,对小鼠体重的增长抑制率可达50％,与rhCNTF每天注射2次的体重增长抑制作用相当。结论：CN10蛋白在PEG化修饰后,其减重效应持续时间明显延长。     

Suramin Induces Phosphorylation of the High-Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet-derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose-dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin-induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine-phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high-affinity NGF (TrkA) receptor. Activation of other members of the signal-transduction cascade (Shc, p21 ^ras , Raf-1, ERK-1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.     

Binding Interactions of Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor with the Different Subunits of Their High Affinity Receptors

Abstract: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low-affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10⁻¹⁰ M , whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.     

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.     

V170 Area Plays an Essential Role in the Biological Activity of Human Ciliary Neurotrophic Factor

Abstract: The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.     

转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

为了实现在体内更持久的药效作用,根据睫状神经营养因子CNTF第17位为游离半胱氨酸残基,而转铁蛋白Tf无游离半胱氨酸的特点,采用N-羟基琥珀酰亚胺-聚乙二醇5K-马来酰亚胺(NHS-PEG5k-MAL)作为偶联剂,实现了两者定点偶联,然后结合蛋白自身特性制定纯化方法,制备获得纯度高于90%的转铁蛋白-聚乙二醇5k-睫状神经营养因子(Tf-PEG5k-CNTF)耦合物。高效凝胶色谱和动态光散射分析显示耦合物的表观分子体积大于两蛋白之和。细胞试验结果显示耦合物的活性下降至原蛋白的65.8%。大鼠药代动力学试验显示Tf-PEG5k-CNTF耦合物在体内的代谢半衰期延长至8.2小时,与CNTF原蛋白相比提高了约17倍。小鼠动物试验显示在每周2次的给药频率,每次1.0 mg/kg的剂量下,Tf-PEG5k-CNTF能更为显著地影响小鼠对食物摄入量和减轻体重。因此,转铁蛋白偶联技术可用于脑部靶向蛋白药物的长效递送。