Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Multiple cholinergic differentiation factors are present in footpad extracts: comparison with known cholinergic factors.

Sweat glands in rat footpads contain a neuronal differentiation activity that switches the phenotype of sympathetic neurons from noradrenergic to cholinergic during normal development in vivo. Extracts of developing and adult sweat glands induce changes in neurotransmitter properties in cultured sympathetic neurons that mimic those observed in vivo. We have characterized further the factors present in the extract and compared their properties to those of known cholinergic factors. When assayed on cultured rat sympathetic neurons, the major activities in footpad extracts from postnatal day 21 rat pups that induce choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP) and reduce catecholamines and neuropeptide Y (NPY) are associated with a soluble protein of 22-26 x 10(3) M(r) and a pI of 5.0. These properties are similar to those of ciliary neurotrophic factor (CNTF). Moreover, the purified fraction from footpads has ciliary neurotrophic activity. Antibodies to CNTF that immunoprecipitate all differentiation activity from sciatic nerve extracts, a rich source of CNTF, immunoprecipitate 80% of the cholinergic activity in the footpad extracts, 50% of the VIP and 20% of the NPY activities. Neither CNTF protein nor CNTF mRNA, however, can be detected in immunoblot and northern analysis of footpads even though both CNTF protein and mRNA are evident in sciatic nerve. CNTF-immunoreactivity is associated with a sparse plexus of sensory fibers in the footpad but not with sweat glands or the Schwann cells associated with them. In addition, in situ hybridization studies with oligonucleotide probes failed to reveal CNTF mRNA in sweat glands. Comparison of the sweat gland differentiation activity with the cholinergic differentiation factor from heart cells (CDF; also known as leukemia inhibitory factor or LIF) suggests that most of the cholinergic activity in foot pads is biochemically distinct from CDF/LIF. Further, antibodies that block the activity of CDF/LIF purified from heart-cell-conditioned medium do not block the ChAT-inducing activity present in footpad extracts of postnatal day 8 animals. A differentiation factor isolated from skeletal muscle did not induce cholinergic properties in sympathetic neuron cultures and therefore is unlikely to be the cholinergic differentiation factor produced by sweat glands. Taken together, our data suggest that there are at least two differentiation molecules present in the extracts and that the major cholinergic activity obtained from footpads is related to, but distinct from, CNTF. The second factor remains to be characterized. In addition, CNTF associated with sensory fibers may make a minor contribution to the cholinergic inducing activity present in the extract.     

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.     

The CNTF/LIF signaling pathway regulates developmental programmed cell death and differentiation of rod precursor cells in the mouse retina in vivo

Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.     

Effects of quercetin combined with anticancer drugs on metastasis-associated factors of gastric cancer cells: in vitro and in vivo studies

Chemotherapy is essential to most patients with gastric cancer and the anticancer drug, irinotecan (CPT-11), and its metabolite, SN-38, an inhibitor of DNA topoisomerase I, are first-line chemotherapies for gastric cancer. Quercetin, a flavonoid that is widely found in various vegetables and fruits, has the ability to potentiate the efficacy of anticancer drugs. The purpose of this study was to investigate the therapeutic effect of quercetin combined with irinotecan/SN-38 in the AGS human gastric cancer cell line in vitro and in vivo. The in vitro study evaluated the efficacy of high-dose SN-38 and quercetin combined with low-dose SN-38 on cell viability, apoptosis, and β-catenin expression. Results showed that cell viability and the percentage of apoptosis in combined treatments with quercetin and SN-38 were comparable to treatment with high-dose SN-38 alone. AGS cells treated with a high dose of SN-38 exhibited up-regulation of β-catenin protein expression, whereas quercetin-treated cells (either quercetin alone or combined with low-dose SN-38) exhibited lower protein levels of β-catenin. In the AGS xenograft mouse model, gene expression of cyclooxygenase-2 and epithelial-mesenchymal transition-related markers, such as Twist1 and ITGβ6, were lower in combined treatments with quercetin and low-dose irinotecan than high-dose irinotecan alone. Furthermore, the concentration of angiogenesis-associated factors (vascular endothelial growth factor (VEGF)-A and VEGF-receptor 2) and percentage of Tie2-expressing monocytes was significantly down-regulated in combined treatments with quercetin and irinotecan. These results suggest that quercetin may enhance the efficacy of irinotecan/SN-38 in the human AGS cell line.     

The Cholinergic Stimulating Effects of Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Are Mediated by Protein Kinase C

Abstract: The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC-depleted sympathetic neurons. The inhibition of CNTF-stimulated ChAT by phorbol ester occurred in a dose-dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate. These observations suggest that a common PKC-dependent pathway, which is independent of phosphatidylinositol 4,5-bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell-cell contact in cultured sympathetic neurons.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha

Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRbeta-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRbeta- or CNTFRalpha-containing receptors.     

Temperature-dependent,neurotrophic factor-elicited,neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33 degrees C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39 degrees C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39 degrees C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33 degrees C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39 degrees C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

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.     

Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways

Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.     

Steroid synthesis by primary human keratinocytes; implications for skin disease

Mechanical stretch has been shown to increase vascular endothelial growth factor (VEGF) expression in cultured myocytes. Sympathetic neurons (SN) also possess the ability to express and secrete VEGF, which is mediated by the NGF/TrkA signaling pathway. Recently, we demonstrated that SN respond to stretch with an upregulation of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF). Whether stretch increases neuronal VEGF expression still remains to be clarified. Therefore, SN from the superior cervical ganglia of neonatal Sprangue Dawley rats were exposed to a gradual increase of stretch from 3% up to 13% within 3 days (3%, 7% and 13%). Under these conditions, the expression and secretion of VEGF was analyzed. Mechanical stretch significantly increased VEGF mRNA and protein expression (mRNA: control = 1 vs. stretch = 3.1; n = 3/protein: control = 1 vs. stretch = 2.7; n = 3). ELISA experiments to asses VEGF content in the cell culture supernatant showed a time and dose dependency in VEGF increment due to stretch. NGF and CNTF neutralization decreased stretch-induced VEGF augmentation in a significant manner. This response was mediated in part by TrkA receptor activation. The stretch-induced VEGF upregulation was accompanied by an increase in HIF-1α expression. KDR levels remained unchanged under conditions of stretch, but showed a significant increase due to NGF neutralization. In summary, SN respond to stretch with an upregulation of VEGF, which is mediated by the NGF/CNTF and TrkA signaling pathway paralleled by HIF-1α expression. NGF signaling seems to play an important role in regulating neuronal KDR expression.