Regulatory mechanisms of nerve growth factor synthesis in vitro

Astroglial cells and various types of non-neuronal cells in the peripheral nervous system, such as epithelial, Schwann and fibroblast cells synthesize and secrete nerve growth factor (NGF) in culture. NGFmRNA contents are well-correlated with the density of axonal projection from NGF-sensitive neurons, suggesting that NGF synthesis in vivo tissues is regulated by neuronal environments. We investigated neuronal regulations of NGF synthesis using cultured mouse astroglial cells and rat pheochromocytoma PC12 cells. It was found that astroglial NGF synthesis was enhanced by the addition of catecholamine into the cultured medium or the co-culture with differentiated PC12 cells. These results suggest that NGF synthesis in the in vivo tissues is increased by the release of catecholamine as neurotransmitters and/or the contact of NGF-producing cells with differentiated cell bodies and neurites of NGF-sensitive neurons.     

Human fibroblast cells synthesize and secrete nerve growth factor in culture.

Using our enzyme immunoassay system developed for recombinant hNGF, we examined the synthesis and secretion of human NGF (hNGF) by human fibroblast (WS-1) cells. The amount of the factor secreted by WS-1 cells increased linearly and a significant amount of NGF was detected in the conditioned medium of WS-1 cultures. WS-1 NGF showed properties identical to those of recombinant human NGF in immunoreactivity and molecular weight. An increase in cell density or the withdrawal of serum from the culture medium caused a drastic decrease in the rate of NGF secretion. These results suggest that WS-1 cells are able to synthesize and secrete hNGF in culture and that the synthesis/secretion is regulated in a growth phase-dependent manner.     

Nerve growth factor promotes development of glucose-induced insulin secretion in rat neonate pancreatic beta cells by modulating calcium channels

Neonatal beta cells are functionally immature as they secrete less insulin than adults and lack of glucose response. The mechanisms that participate in the functional maturation of these cells are not known. Adult rat beta cells synthesize and secrete nerve growth factor (NGF) and express NGF receptors. NGF increases glucose-induced insulin secretion by modulating electrical activity in adult beta cells. In this work, we explored if NGF is involved in the maturation of glucose-induced insulin secretion coupling in rat neonate beta-cells.     

Remodelling sympathetic innervation in rat pancreatic islets ontogeny

Background  

Pancreatic islets are not fully developed at birth and it is not clear how they are vascularised and innervated. Nerve Growth Factor (NGF) is required to guide sympathetic neurons that innervate peripheral organs and also in cardiovascular system and ovary angiogenesis. Pancreatic beta cells of a transgenic mouse that over-expressed NGF in attracts sympathetic hyper-innervation towards them. Moreover, we have previously demonstrated that adult beta cells synthesize and secrete NGF; however, we do not know how is NGF secreted during development, nor if it might be trophic for sympathetic innervation and survival in the pancreas.     

Neurotrophic factor-like activity in Drosophila.

The NGF-family of neurotrophic factors are structurally similar peptides with related functional properties. So far, this family of neurotrophic factors has only been identified in the vertebrate nervous system. We have determined that cultured Drosophila embryonic cells produce and secrete into medium, an activity which stimulates neurite outgrowth of embryonic chick sensory ganglia. This Drosophila activity can be blocked by antibodies to mouse NGF, indicating an immunological relationship between the Drosophila factor, mouse NGF and possibly other vertebrate neurotrophic factors. Addition of mouse NGF to Drosophila embryonic cells in culture results in increased cell number and enrichment of the neuronal phenotype, indicating that Drosophila cells have the ability to respond to the vertebrate factor. In addition, poly(A)+RNA extracted from Drosophila contains a single 1.4 Kb band which cross-hybridizes with a mouse NGF cRNA probe. These results indicate that vertebrate neurotrophic factor-like functions may operate in a genetically defined invertebrate species.     

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.     

L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC12 pheochromocytoma cells

Two mouse cell lines, 3T3 and L, reported to secrete an NGF-like activity in the culture medium were co-cultured with the pheochromocytoma cell line PC12 which responds to NGF in vitro. In these co-cultures mitomycin-treated L or 3T3 cells were employed at low cell density (1 000 cells/cm2). L cells, but not 3T3, promoted efficiently neurite outgrowth of PC12. The response of the PC12 cells was blocked by an antiserum to male mouse submaxillary gland beta NGF. The NGF secreted by the L cells and immunoprecipitated by this antiserum co-migrated with the submaxillary gland beta NGF monomer in SDS-polyacrylamide gels. Surprisingly the neurite-promoting activity of media conditioned by L or by L-PC12 co-cultures was at most one-tenth of that expected on the basis of the response of PC12 cells in the co-cultures. This was not due to proteolytic degradation of the NGF-like factor or to losses by manipulation of the media. It seems therefore that co-cultures provide conditions which enhance the effect of the factor. Possible mechanisms responsible for this effect are discussed.     

Secretion of nerve growth factor by central nervous system glioma cells in culture

Bacteriophage immunoassays, radioimmunoassays, and biological assays have been used to measure levels of NGF in media conditioned by rat C-6 glioma cells in culture. By all three criteria, these cells secrete a macromolecule which is indistinguishable from mouse submandibular gland NGF.     

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.     

Synthesis of immunoglobulin by substrate attached mouse myeloma cells

Cultured mouse myeloma cells grow in suspension and synthesize and secrete large amounts of immunoglobulin. Mouse myeloma cells which attach to a plastic substratum have been obtained by mutagenesis and subsequent selection. Normal mouse myeloma cells will also attach to plastic tissue culture dishes pre-treated with poly-L-lysine. The attached cells synthesize and secrete the same large amounts of immunoglobulin as the suspended cells.     

Sympathetic neurons synthesize and secrete pro‐nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF‐immunoreactive proteins synthesized by cultured NGF‐dependent and ‐independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro‐NGF protein. These findings suggest that a potential NGF‐sympathetic neuron autocrine loop may exist in this prototypic target‐dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival. © 2003 Wiley Periodicals, Inc. J Neurobiol 38–53, 2003     

Structural analysis of fibrinogen synthesized by cultured chicken hepatocytes in the presence or absence of dexamethasone

Hepatocyte monolayers, derived from chick embryos and cultured in chemically defined medium without hormones, synthesize and secrete fibrinogen that resembles chicken plasma fibrinogen immunochemically and structurally. Addition of a synthetic glucocorticoid, dexamethasone, to the cultured cells resulted in an appreciable and relatively selective increase in fibrinogen synthesis. Autoradiography of fibrinogen that had been metabolically labelled with [35S]methionine and then subjected to SDS-polyacrylamide gel electrophoresis, unreduced or under disulfide-reducing conditions, revealed that only dimeric forms of fibrinogen, containing undegraded A alpha, B beta, and gamma chains, were secreted under stimulated and unstimulated culture conditions.     

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.     

beta-Nerve growth factor (beta NGF) receptors on glial cells. Cell-cell interaction between neurones and Schwann cells in cultures of chick sensory ganglia.

Receptors for beta-nerve growth factor (beta NGF), so far regarded as specific cell surface markers of certain peripheral neurones, were found to be expressed on cultured non-neuronal cells of chick embryo dorsal root ganglia (drg) (Kd beta NGF = 2 X 10(-9) M). Autoradiography revealed that binding of [125I] beta NGF was restricted to a subpopulation of the non-neuronal drg cells. Cultured embryonic skin fibroblasts, liver cells, gut cells, muscle fibroblasts, myoblasts, and myotubes, as well as macrophages and the cell lines 3T3, 3T3SV40, BHK, BHK Py, PCC3 and ND1, did not express receptors for beta NGF. Non-neuronal drg cells obtained by a procedure designed for the preparation of pure Schwann cells, as well as RN6 Schwannoma cells, were beta NGF receptor positive. The beta NGF receptor-positive non-neuronal drg cells displayed behaviour typical of Schwann cells in their interaction with drg neurones in single cell, as well as explant cultures. Three stages of neurone-Schwann cell interaction were discernible: (1) association--neurites preferentially grew over beta NGF receptor-positive non-neuronal cells; (2) cell division/alignment--beta NGF receptor-positive non-neuronal cells were induced to proliferate and aligned and elongated along neurites; (3) ensheathment--the outline of beta NGF receptor-positive non-neuronal cells and neurites merged. In drg cell cultures prepared from embryonic stages E6-E10, 25-40% of the non-neuronal cells were beta NGF receptor-positive. Later in development, from E12 onward, less than or equal to 1% of the cultured non-neuronal cells expressed beta NGF receptors.     

Regulation of nerve growth factor synthesis/secretion by catecholamine in cultured mouse astroglial cells

The nerve growth factor (NGF) synthesis/secretion by cultured mouse astroglial cells was modulated by catecholamine. In quiescent cells, epinephrine (EN) and dopamine (DA) markedly increased the NGF content in the conditioned medium (CM). Conversely, EN, DA, and norepinephrine (NE) decreased the NGF content in growing cells. Cholinergic agonists, metacholine and carbamylcholine, slightly increased the NGF content in quiescent cells, but showed no effects on growing cells. Other neurotransmitters tested had no effects on either growing or quiescent cells. These results suggest that catecholamine is one of the molecules responsible for regulation of NGF synthesis/secretion in the mouse brain.     

In vivo activated brain astrocytes may produce and secrete nerve growth factor-like molecules.

The cellular localization of the nerve growth factor-like immunoreactivity (NGF-LIR) has been studied in the septum and hippocampus of the rat brain 7 days following partial electrolytic lesion (2 mA, 30 s) of the septohippocampal pathways or after single intraventricular administration of 15 U of interleukin-1 beta (IL-1 beta). A double immunostaining technique which allowed a simultaneous localization of NGF-LIR and that of astroglia marker glial fibrillary acidic protein was used. Our data show that after both treatments, apart from neuronal localization of NGF-LIR typical for normal brain, many astrocytes both in the septum and hippocampus became NGF-like immunoreactive. Besides, NGF-LIR often formed a "halo" reaction around astrocytes. These results support the notion that activated in vivo brain astrocytes may, just as astrocytes growing in vitro, synthesize and secrete NGF-like molecules. Our findings may be of importance in considerations concerning trophic support to the cholinergic neurons of the basal forebrain nuclei whose impaired function is essentially responsible for some cognitive deficits in neurodegenerative diseases such as Alzheimer disease.     

Effects of NGF and glucocorticoid on NGF receptor immunolabeling of cultured rhesus adrenal chromaffin cells

Nerve growth factor (NGF) promotes the outgrowth of neurites from cultured adrenal chromaffin cells from adult rhesus monkeys, but little is known about the distribution, at the cellular level, of the NGF receptors (NGFR) responsible for this response. We examined changes in immunostaining for NGFR in chromaffin cells cultured for 4 weeks in the presence or absence of NGF, with or without dexamethasone (DEX), which inhibits neuritic outgrowth from these cells. Purified cultures of adrenal chromaffin cells from adult rhesus monkeys were grown for up to 9 weeks in NGF, DEX, NGF plus DEX, or control medium. Cells were immunolabeled with three different monoclonal antibodies directed against different epitopes of the human NGFR. Although the distribution of immunolabeling was not uniform from cell to cell, the overall intensity of NGFR immunolabeling varied dramatically between different growth conditions. Of greatest interest, DEX-treated cells stained the most intensely at all time points, while the intensity of immunolabeling was much fainter in NGF-treated cells and decreased with time in culture. In contrast to the intensity of labeling, the proportion of chromaffin cells with immunoreactivity increased with time in all treatment groups. Thus, GCs do not appear to antagonize the effects of NGF merely by decreasing the total number of immunoreactive NGFR on the surface of these cells.     

Casein secretion by mammary gland epithelia from collagen gel cultures and lactating glands

Amino acid incorporation experiments show that epithelial cells from lactating mouse mammary glands and from collagen gel culture both synthesize and secrete four principal phosphocaseins (p45, p40, p27, and p23 kD). In both cases, however, the casein production is largely dominated by the p27 species. The average percentage distribution of the above casein species in medium from cultured epithelia is approximately 13%, 6%, 68%, and 14%, respectively; for milk the distribution is approximately 23%, 7%, 54%, and 16%. The predominance of the p27 species is not a consequence of extensive extracellular differential degradation of the secreted caseins since no significant casein degradation was observed in culture medium, either in contact or isolated from epithelial cell monolayers. Synthesis and secretion of all the caseins by cultured epithelia is dependent upon insulin, prolactin, and hydrocortisone. Presumably some intracellular events result in the secretion of p27 as the principal casein in mouse milk. Apparently, some selection factor(s) operate to make p27 a major nitrogenous nutritional component for a newborn mouse. In addition, on a quantitative basis, the relative levels of various caseins secreted by epithelia from lactating mammary glands is essentially duplicated by epithelia in collagen gel culture.     

Endorphin-like immunoreactivities in uncultured and cultured human peripheral blood mononuclear cells.

It has been reported that cells of the immune system produce and release considerable amounts of pro-opiomelanocortin (POMC) -derived peptides in response to coculture with a variety of stimulatory agents. The present study investigated whether extracts of human peripheral blood mononuclear cells (PBMC) contain immunoreactivity for beta-endorphin (beta E) and related peptides. Using four endorphin RIA systems with different specificities, extracts of freshly isolated PBMC and PBMC cultured in the presence or absence of mitogens or of corticotropin releasing factor (CRF) and vasopressin (VP), were analyzed. With a radioimmunoassay (RIA) system directed to the midportion of beta E, immunoreactivity (MP beta E-IR) was readily detectable, although the concentration was extremely low (ca. 200 pg/10(7) cells). beta E immunoreactivity (beta E-IR) and alpha-endorphin immunoreactivity (alpha E-IR), as determined in C-terminally directed RIA systems, were present in even lower concentrations. gamma-Endorphin immunoreactivity (gamma E-IR) was hardly detectable. Of subsets enriched in T-cells, B-cells or monocytes, the highest concentration of MP beta E-IR was detected in extracts of monocytes. Coculture of PBMC with the mitogen Concanavalin A (Con A) or Phytohaemagglutinin (PHA) increased the amount of MP beta E-IR in extracts of the cells. No increase in alpha E-IR, however, was detected, whereas beta E-IR was only increased in extracts of cells cultured in the presence of Con A. No increase, in any of the immunoreactivities, was observed in extracts of PBMC cultured with bacterial lipopolysaccharide (LPS) or with the combination of CRF and VP, both stimuli that have been reported to induce POMC peptides in cultured PBMC. The present data show that human PBMC contain endorphin-like immunoreactivity, but in very small amounts. The extremely low concentrations and the ineffectiveness of LPS and the combination of CRF and VP to increase the endorphin-like immunoreactivity raise questions about the reported capacity of PBMC to synthesize POMC-derived peptides.