Increased Nerve Growth Factor Secretion Research Articles

Promotion effect of the propolis from Jeju Island, Korea, on NGF secretion in human glioblastoma cells.

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees (Apis mellifera). We previously found that propolis collected on Jeju Island, located off the southern coast of Korea, originates from a single plant, Angelica keiskei KOIDZUMI (Ashitaba). A. keiskei has been well-studied as a health food and has been reported to promote nerve growth factor (NGF) production. Propolis formed from the resin of A. keiskei is expected to have a similar promotional effect on NGF production. NGF is a potential pharmacological agent for Alzheimer's disease. In this study, the effects of an ethanolic extract of propolis from Jeju Island (EEPJ) on NGF secretion and cell viability in T98G human glioblastoma cells were evaluated. Ethanolic extracts of propolis from Brazil (Baccharis type) and from Uruguay (Populus type) were also studied for comparison. We found that EEPJ significantly increased NGF secretion in the cells in a concentration-dependent manner. Furthermore, the effects of 27 compounds previously isolated from EEPJ were also evaluated. Several compounds were found to have a promotion effect on NGF secretion, and the structure-activity relationships of the compounds were considered relative to their promotional effect on NGF biosynthesis. The promotional effect of EEPJ is a characteristic biological activity that is not present with other propolis types, so the propolis from Jeju Island may have potential applications as a therapeutic candidate for Alzheimer's disease.

Asparagus cochinchinensis stimulates release of nerve growth factor and abrogates oxidative stress in the Tg2576 model for Alzheimer\u2019s disease

BackgroudUse of multifunctional drugs with neurotrophic supporting and oxidative stress suppressing activity may be considered a therapeutic strategy to protect or repair cellular damage caused during the progression of Alzheimer’s disease (AD). In this study, we investigated the therapeutic effects of aqueous extract of A. cochinchinesis root (AEAC), particularly its role as a nerve growth factor (NGF) stimulator and anti-oxidant in Tg2576 mice showing AD phenotypes of human.MethodsTg2576 mice were received 100 mg/kg/day AEAC via oral administration, while mice in the Vehicle treated group received dH2O for 4 weeks. Non-Tg littermates were used as a control group. Following AEAC treatment for 4 weeks, NGF function, anti-oxidantive status, Aβ-42 peptide level, γ-secretase expression and neuronal cell functions were analyzed in the brain of Tg2576 mice.ResultsAEAC containing flavonoids, phenols, saponins and protodioscin induced enhancement of NGF secretion and decreased intracellular ROS in the neuronal and microglial cell line. These effects as well as enhanced SOD levels were also detected in AEAC treated Tg2576 mice. The expression of p-Akt among downstream effectors of the high affinity NGF receptor was dramatically recovered in AEAC treated Tg2576 mice, while the expression of p75NTR was slightly recovered in the same group. Significant recovery on the level of Aβ-42 peptides and the expression of γ-secretase members including PS-2, APH-1 and NCT were detected in AEAC treated Tg2576 mice. Furthermore, AEAC treated Tg2576 mice showed decreased numbers of dead cells and suppressed acetyl choline esterase (AChE) activity.ConclusionsThese results suggest that AEAC contribute to improving the deposition of Aβ-42 peptides and neuronal cell injuries during the pathological progression stage of AD in the brain of Tg2576 mice through increased NGF secretion and suppressed oxidative stress.

Let-7 microRNAs Regenerate Peripheral Nerve Regeneration by Targeting Nerve Growth Factor

Peripheral nerve injury is a common clinical problem. Nerve growth factor (NGF) promotes peripheral nerve regeneration, but its clinical applications are limited by several constraints. In this study, we found that the time-dependent expression profiles of eight let-7 family members in the injured nerve after sciatic nerve injury were roughly similar to each other. Let-7 microRNAs (miRNAs) significantly reduced cell proliferation and migration of primary Schwann cells (SCs) by directly targeting NGF and suppressing its protein translation. Following sciatic nerve injury, the temporal change in let-7 miRNA expression was negatively correlated with that in NGF expression. Inhibition of let-7 miRNAs increased NGF secretion by primary cultured SCs and enhanced axonal outgrowth from a coculture of primary SCs and dorsal root gangalion neurons. In vivo tests indicated that let-7 inhibition promoted SCs migration and axon outgrowth within a regenerative microenvironment. In addition, the inhibitory effect of let-7 miRNAs on SCs apoptosis might serve as an early stress response to nerve injury, but this effect seemed to be not mediated through a NGF-dependent pathway. Collectively, our results provide a new insight into let-7 miRNA regulation of peripheral nerve regeneration and suggest a potential therapy for repair of peripheral nerve injury.

Nicotine stimulates nerve growth factor in lung fibroblasts through an NFκB-dependent mechanism.

RationaleAirway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment.MethodsPrimary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid.ResultsNGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells.ConclusionNicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.

Precautionary effects of RedLiriope platyphyllaon NGF secretion and Aβ42 deposition under the preclinical stage of Alzheimer's disease in Tg2576 mice

Red Liriope platyphylla (RLP) has been manufactured from Liriope platyphylla (L. platyphylla, LP) roots using steaming process and investigated as a curative agent for treatment of diabetes, obesity and neurodegenerative disorders. To examine the precautionary effects of aqueous extract RLP (AEtRLP) on the preclinical stages of Alzheimer's Disease (AD), alterations of the key factors influencing AD were investigated in Tg2576 mice after AEtRLP7 treatment for 4 months. Aβ-42 peptides level was significantly decreased in the brain of AEtRLP7-treated Tg2576 mice compared to vehicle-treated Tg2576 mice, although significant differences on improving behavioral defects were not observed in the same group. The concentration of nerve growth factor (NGF) in serum was also higher in AEtRLP7-treated Tg2576 mice than vehicle-treated Tg2576 mice. However, the phosphorylation of TrkA and Erk among the downstream effectors of the high affinity NGF receptor was significantly lower in AEtRLP7-treated Tg2576 mice. A similar pattern was observed in the expression level of downstream effectors within low affinity NGF receptor. Overall, these results suggest that AEtRLP7 can contribute to preventing the production and deposition of Aβ-42 peptides during the early progression stage of AD in the brain of Tg2576 mice through increased NGF secretion.

Astrocytes and Chronic Pain Mechanisms—The Role of Histamine, IL-1β and NGF

ABSTRACTIn exaggerated pain states, the activated astrocytes release several signalling molecules involved in cellular mechanisms of chronic pain. Among them, nerve growth factor (NGF) and interleukin (IL)-1β are both recognized as potent algogens. We previously showed that histamine is a potent stimulator of NGF production in rat cortical astrocytes in primary culture. Since histamine and IL-1β have common interactions in different physiological responses, in the present work we were interested to elucidate the molecular mechanisms involved in the interactions between histamine, IL-1β and NGF that could contribute to the processing of chronic pain. As an experimental model we used cultured rat cortical astrocytes. NGF and IL-1β levels in the culture medium were measured by ELISA. IL-1β mRNA expression was determined by RT PCR.The results showed that the co-treatment of the cultured astrocytes with histamine and IL-1β significantly increased NGF secretion in comparison to the secretion observed with either histamine, or IL-1β alone. The histamine and IL-1β effect on NGF secretion was additive, dose-dependent and increased with increased concentrations of either histamine, or IL-1β. The additive effect of histamine and IL-1β on NGF secretion was strongly suppressed by histamine H1 receptor antagonist/inverse agonist mepyramine, protein kinase C (PKC) inhibitors (GF 109203X, Go 6976), and mitogen-activated protein kinase kinase (MAPK)-1 inhibitor (PD 98059); however, they did not influence significantly the NGF secretion evoked by IL-1β alone. Histamine also stimulated the secretion of IL-1β from cultured astrocytes and induced higher expression of IL-1β mRNA in comparison to untreated cells.We concluded that histamine potently interacts with the synthesis and secretion of IL-1β and NGF in astroglial cells, and therefore can contribute to the development and maintenance of chronic pain, mediated by both algogens.

Complexity of Astrocyte-Motor Neuron Interactions in Amyotrophic Lateral Sclerosis

Neurons and surrounding glial cells compose a highly specialized functional unit. In amyotrophic lateral sclerosis (ALS) astrocytes interact with motor neurons in a complex manner to modulate neuronal survival. Experiments using chimeric mice expressing ALS-linked mutations to Cu,Zn superoxide dismutase (SOD-1) suggest a critical modulation exerted by neighboring non-neuronal cell types on disease phenotype. When perturbed by primary neuronal damage, e.g. expression of SOD-1 mutations, neurons can signal astrocytes to proliferate and become reactive. Fibroblast growth factor-1 (FGF-1) can be released by motor neurons in response to damage to induce astrocyte activation by signaling through the receptor FGFR1. FGF-1 stimulates nerve growth factor (NGF) expression and secretion, as well as activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Nrf2 leads to the expression of antioxidant and cytoprotective enzymes such as heme oxygenase-1 and a group of enzymes involved in glutathione metabolism that prevent motor neuron degeneration. However, prolonged stimulation with FGF-1 or SOD-mediated oxidative stress in astrocytes may disrupt the normal neuron-glia interactions and lead to progressive neuronal degeneration. The re-expression of p75 neurotrophin receptor and neuronal NOS in motor neurons in parallel with increased NGF secretion by reactive astrocytes may be a mechanism to eliminate critically damaged neurons. Consequently, astrocyte activation in ALS may have a complex pathogenic role.

Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures

We found that prostaglandin (PG) D2 and PGE2, which are major PGs in the brain of mammals, powerfully induced the secretion of nerve growth factor (NGF) from cultured mouse astrocytes; PGE2 or PGD2 induced an approximately 12- or 19-fold increase in NGF secretion after a 24-h incubation, respectively. Moreover, it was found that the sequential metabolites of PGD2, PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2, induced the NGF secretion to the culture medium strikingly (60–98-fold of the control after a 24-h incubation). NGF secretion induced by the J2 series of PGs was accompanied by the increased expression of NGF mRNA. These PGs also stimulated the secretion/synthesis of brain-derived neurotrophic factor (BDNF). Our findings suggest that PGs play a neuroprotective role by inducing NGF and BDNF production in the central nervous system.

Preovulatory rise of NGF in ovine follicular fluid: possible involvement in the control of oocyte maturation.

Since nerve growth factor (NGF) is produced in vitro by granulosa cells after gonadotropin stimulation, the present research has been designed to investigate whether this neurotropin is involved in the events triggered by the gonadotropin surge that lead the follicle to ovulate a mature oocyte. To this aim, NGF levels in follicular fluid, collected before or 20 hours after the gonadotropin surge, was measured by ELISA. To evaluate whether NGF may have a non-neurotropic effect on follicle cells, the presence of NGF receptors was investigated by immunohistochemistry and further evaluated by analysing the tyrosine-phosphorylation pattern after NGF stimulation in vitro. The effect of NGF on the degree of cumulus expansion, cumulus-oocyte metabolic coupling, and meiotic maturation was finally studied by using the culture of follicle-enclosed oocyte. The results demonstrate that GnRH causes a dramatic rise of NGF in large follicles. Immunohistochemistry revealed a discrete positivity for trkA receptors localised in cumulus cells. Tyrosine phosphorylation pattern confirms that somatic cells are capable to transduce NGF signal. By contrast, all the oocytes examined were negative for trkA and did not change the phosphorylation pattern after NGF. In vitro NGF (100 ng/ml) induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling similar to that produced by gonadotropins. The addition of NGF also caused the resumption of meiosis in more than 70% of the oocytes analysed with an effect that is only slightly less pronounced than that of gonadotropins (80%). The increase in NGF secretion following gonadotropin surge suggests that this neurotropin may be involved in the control of oocyte maturation.

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1β (IL-1β). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1α and IL-1β) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [125I] IL-1β to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1α (Ki of 341.1 pM) and IL-1β (Ki 59.9 pM), as well as the antagonist IL-1ra (Ki 257.6 pM), displaced specific [125I] IL-1β binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [125I] IL-1β binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1α and IL-1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1β (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1α by itself had no stimulatory action on NGF release. The simultaneous application of IL-1α and IL-1β elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1β. We concluded that IL-1β-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1α and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.

Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes.

Interactions involved in the regulation of nerve growth factor (NGF) release by inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) were examined in rat neonatal cortical astrocytes in primary culture. Abstract Exposure of cultured astrocytes to IL-1β, IL-6, and TGF-β1 resulted in the stimulation of NGF secretion. Treatment of cells for 24 h with IL-1β (10 U/ml), IL-6 (5 ng/ml) and TGF-β1 (5 ng/ml) caused 3-, 1.8-, and 2.8-fold increase in NGF secretion as compared to the control cells. In contrast, TNF-α (30 ng/ml) by itself had no stimulatory action on NGF release whereas co-stimulation of astrocytes with IL-1β and TNF-α showed a synergistic interaction. Co-treatment of astrocytes with IL-1β and TGF-β1 increased NGF secretion in an additive way, whereas simultaneous application of IL-1β and IL-6 resulted in the inhibitory effect on NGF secretion. Our results suggest that interactions between cytokines used cause the stimulation of NGF secretion through different regulatory mechanisms.

Increased nerve growth factor mRNA stability may underlie elevated nerve growth factor secretion from hypertensive vascular smooth muscle cells

Altered nerve growth factor (NGF) regulation has been linked to the pathophysiology of hypertension. Vascular smooth muscle cells from an inbred hypertensive, but normoactive rat strain (WKHT) secreted NGF at a greater rate than from a hyperactive, normotensive strain (WKHA). Exposure to phorbol ester increased NGF secretion rates from WKHT by 400–800% but not from WKHA vascular muscle. NGF secretion rates from both WKHT and WKHA vascular cells were elevated by co-application of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) by 300–1000%. This response was partially attenuated by actinomycin D, an inhibitor of RNA transcription. These results suggest that regulation of NGF production does not occur solely at the level of transcription and post-transcriptional mechanisms operate. Analysis of NGF mRNA stability in the two strains following PDGF and TGF-β1 treatment showed that NGF mRNA in WKHT had a half-life of 126.2±11.68 min while in WKHA vascular smooth muscle cells, the half-life was 47.33±11.98 min. In addition to increased NGF mRNA stability in WKHT vascular muscle, these cells have an increased translational efficiency of NGF protein; elevated synthesis of NGF protein per unit NGF mRNA. Differences in signaling pathways may result in increased NGF mRNA stability and translational efficiency that may account for the elevated NGF protein in WKHT vascular smooth muscle cells.

Thrombin regulates nerve growth factor secretion from vascular, but not bladder smooth muscle cells.

The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet. We have been examining the cellular processes of NGF delivery and secretion in smooth muscle. NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media. Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied. The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs. The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required. The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C. These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle. Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult.

Synergistic increase in nerve growth factor secretion by cultured vascular smooth muscle cells treated with injury-related growth factors

Vascular smooth muscle (VSM) cells comprise one of the primary targets of the sympathetic nervous system and have been shown to secrete nerve growth factor (NGF). There is increasing evidence that changes in the levels of NGF in the adult may underlie certain pathological conditions. To investigate the potential role of altered NGF production in vascular disease, VSM cell cultures were treated with injury-related growth factors and the culture medium was assayed for NGF using a two-site enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (PDGF), a potent VSM mitogen, caused a dose-dependent increase in NGF secretion. After 4 hr, PDGF-treated cultures contained 10 times more NGF than control cultures. NGF release remained elevated for 48 hr, but the peak secretion occurred in the first 12 hr after treatment. Transforming growth factor beta (TGF-beta) caused a fivefold increase in NGF at 4 hr when added alone, but synergized with PDGF yielding approximately 50 times more NGF than control cultures. TGF-beta and epidermal growth factor (EGF) also displayed synergism. In contrast, basic fibroblast growth factor (bFGF), which had a modest effect alone, appeared to be additive with TGF-beta. Similarly, interleukin 1-beta (IL-1 beta), which mediates increased NGF synthesis in sciatic nerve lesions (Lindholm et al.: Nature 330:658-659, 1987), showed no synergism with TGF-beta.

Synergistic increase in nerve growth factor secretion by cultured vascular smooth muscle cells treated with injury‐related growth factors

Vascular smooth muscle (VSM) cells comprise one of the primary targets of the sympathetic nervous system and have been shown to secrete nerve growth factor (NGF). There is increasing evidence that changes in the levels of NGF in the adult may underlie certain pathological conditions. To investigate the potential role of altered NGF production in vascular disease, VSM cell cultures were treated with injury-related growth factors and the culture medium was assayed for NGF using a two-site enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (PDGF), a potent VSM mitogen, caused a dose-dependent increase in NGF secretion. After 4 hr, PDGF-treated cultures contained 10 times more NGF than control cultures. NGF release remained elevated for 48 hr, but the peak secretion occurred in the first 12 hr after treatment. Transforming growth factor beta (TGF-β) caused a fivefold increase in NGF at 4 hr when added alone, but synergized with PDGF yielding approximately 50 times more NGF than control cultures. TGF-β and epidermal growth factor (EGF) also displayed synergism. In contrast, basic fibroblast growth factor (bFGF), which had a modest effect alone, appeared to be additive with TGF-β. Similarly, interleukin 1-beta (IL-1β), which mediates increased NGF synthesis in sciatic nerve lesions (Lindholm et al.: Nature 330:658–659, 1987), showed no synergism with TGF-β. J. Neurosci. Res. 47:277–286, 1997. © 1997 Wiley-Liss, Inc.

Altered signalling in vascular smooth muscle from spontaneously hypertensive rats may link medial hypertrophy, vessel hyperinnervation and elevated nerve growth factor.

1. Secretion of nerve growth factor (NGF) by cultured vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto (WKY) strain was measured via two site immunoassay (ELISA). 2. Basal NGF secretion rates of quiescent SHR VSMC in serum-free culture medium were elevated compared to similar WKY VSMC. 3. SHR VSMC displayed increased NGF secretion in response to activation of sympathetic neurotransmitter receptors while VSMC of WKY were largely unresponsive to the agents (phenylephrine, isoproterenol, alpha-beta-methyl ATP, neuropeptide Y). 4. Mitogenic stimulation with platelet-derived growth factor (PDGF) raised SHR NGF secretion rates almost three times more than PDGF increased WKY secretion. 5. SHR VSMC also failed to demonstrate normal inhibitory control over NGF secretion seen in WKY and previously in Sprague-Dawley and Wistar strain VSMC with adenylate cyclase activation and down-regulation of protein kinase C. High concentrations of forskolin stimulated, instead of inhibiting, secretion in SHR. Stimulation was also seen after pretreatment with phorbol ester for 24 h while this inhibited secretion in the WKY. 6. These results confirm that the SHR VSMC are hyperresponsive to growth stimuli such as contractile agonists and mitogens. This hyperresponsiveness includes an abnormal control over NGF secretion such that normally inhibitory treatments stimulate NGF output in the SHR. 7. Because the SHR demonstrates important defects in the major intracellular growth-signalling systems that also regulate NGF output and vessel innervation, the predicted result of the defects is a destructive feed-forward cycle of growth and innervation. This is the SHR phenotype in vivo.

Platelet activating factor induces nerve growth factor production by rat astrocytes

During the course of brain injury and inflammation there is an enhanced secretion of neurotrophic substances. We have examined the effect of platelet activating factor (PAF) , which has been associated with various pathological conditions in the central nervous system, on the secretion of nerve growth factor (NGF) by astrocytes. Enriched cultures of rat cortical astrocytes were treated with various concentrations of PAF for different periods of time. PAF induced a dose-dependent increase in NGF secretion, the maximal increase of 120% obtained with 50 nM PAF after 48 h of treatment. This effect was blocked by the specific antagonist WEB 2086 and was not related to a proliferative response of the cells to PAR Concomitant treatment of the cells with PAF and tumor necrosis factor-α resulted in a synergistic increase in NGF secretion. These results indicate that PAF may provide a neurotrophic signal to injured neurons and may cooperate with TNF-α in this effect.

Fibroblast growth factors stimulate nerve growth factor synthesis and secretion by astrocytes

Nerve growth factor (NGF) is produced and secreted by astrocytes and fibroblasts, but not by microglia, in a primary non-neuronal cell culture derived from newborn rat brains under a standard culture condition. NGF secretion by astrocytes was highest just after passage and then gradually decreased. There is no significant difference in NGF secretion by astrocytes from five sites of origin tested: cerebral cortex, striatum, hippocampus, septum, and cerebrllum. Acidic and basic fibroblast growth factors (aFGF and bFGF) significantly increased NGF secretion by astrocytes. The effect of a FGG was greater than of the of FGF, and the effect of both FGFs ws not additive at the maximu m connentration. The peak of NGF secretion stimulated by aFGF ocuurred 3–12 h after the addition of aFGF. On the other hand, the dramatic increase in cell numbers was observed 12–48 h after stimulation, and the morphological change became significant 24 h after aFGF astimulation. NGF synthetized by astrocytes is rapidly secreted into the culture medium and aFGF enhances NGF secretion from the transcription level, because cycloheximide and actinomycin-D completely inhibited NGF secretion by astrocytes in the presence or absence of aFGF. Epidermal growth factor (EGF), interleukin-1β (IL-1β), and tumor necrosis factor-α (DNF-α) also increased NGF secretion by astrocytes to a certain extent. NGF secretion by astrocytes in the presence of a maximum dose of a FGF was enhanced by the addition of IL-1β or TNF-α, but not EGF. However, platelet-derived growth factor, interleukin-3, and interleukin-6 had no significant effects. FGFs also enhanced NGF secretion by fibroblasts derived from meninges, but not by microglia.

Induction of Nerve Growth Factor Gene Expression by 12‐O‐Tetradecanoyl Phorbol 13‐Acetate

Nerve injury leads to activation of fibroblasts, including stimulation of nerve growth factor (NGF) gene expression. Although interleukin-1 has been implicated as a mediator of NGF gene induction, the underlying mechanisms are not known. We investigated whether 12-O-tetradecanoyl phorbol 13-acetate (TPA), also a known stimulator of protein kinase C, regulates NGF gene expression. We show here that TPA stimulates NGF mRNA in mouse kidney and L929 fibroblasts but not in dispersed salivary cells. NGF mRNA stimulation in L929 cells is delayed by 2 h, is transient, and is followed by a parallel increase in NGF secretion. The induction of NGF mRNA is inhibited by cycloheximide, NGF mRNA levels decrease to similar values after 4 h of incubation with actinomycin D alone or in combination with TPA. These results indicate that the TPA response is cell specific and suggest that it is mediated at the transcriptional level via newly synthesized protein.