β-adrenergic receptor regulation,through cyclic AMP,of nerve growth factor expression in rat cortical and cerebellar astrocytes

1. Type 1 astrocytes prepared from 3-day rat cortex and cerebellum express the 1.3-kb nerve growth factor (NGF) mRNA and synthesize and release beta-NGF. 2. Isoproterenol (IP), a beta-adrenergic agonist, stimulates NGF mRNA content in cortical astrocytes; this increase is blocked by the beta-adrenergic antagonist propranolol but not the alpha-antagonist phenoxybenzamine. The EC50 for the effect of IP is 5 nM. 3. IP increases astrocyte cyclic AMP as does forskolin, which directly activates adenylate cyclase and also increases NGF mRNA content. Cerebellar astrocytes contain about one-third as much NGF mRNA, which can also be increased by forskolin and cyclic AMP. 4. These results suggest that CNS astrocytes can serve as a source of NGF and that the NGF gene is one of the class of cyclic AMP regulated genes.     

HIV-1-infected macrophages induce astrogliosis by SDF-1alpha and matrix metalloproteinases

Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1alpha or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1alpha production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1alpha was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1alpha and MMP production, which implies a mechanism of astrogliosis in HAD.     

Fibroblast growth factor pretreatment reduces epidermal growth factor-induced proliferation in rat astrocytes

Epidermal Growth Factor (EGF) is mitogenic for purified rat astrocytes in primary tissue culture. A combined concomitant treatment by EGF and Fibroblast Growth Factor (FGF) does not reduce the proliferation effect of EGF, however when the astrocytes are pretreated with FGF, their response to an EGF stimulation is reduced by 70%. This inhibition of EGF stimulation by FGF pretreatment is consistent across the EGF dose response curve and perhaps represents a mechanism for local modulation of astrocyte mitogenic effects.     

Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages

Astrocytes have been shown to release an interleukin 3 (IL 3)-like factor that induces the expression of 20-alpha-hydroxysteroid-dehydrogenase (20-alpha SDH) in nu/nu spleen cells, and the proliferation of the IL 3-dependent cell line 32DCL. We have investigated whether astrocyte-derived IL 3 supports growth of macrophages and their representatives in the brain, the microglia cells. Evidence for intercellular communication between murine astrocytes and macrophages became already detectable in co-culture experiments: astrocytes activated with endotoxin resulted in an increased growth of peritoneal macrophages on the astrocyte monolayer. Biochemical analysis of supernatants of activated astrocytes revealed that the IL 3-like factor that stimulated 32DCL cells and the expression of 20 alpha SDH also served as a growth factor for cultured peritoneal macrophages. The same results were obtained by using microglia cells isolated from primary brain cell cultures of newborn mice, which are characterized by their positive reaction for macrophage markers such as Mac-1 and nonspecific esterase. If secreted by reactive astrocytes in vivo, the IL 3-like factor may contribute to the accumulation of macrophages and microglia cells detected in brain lesions of patients with multiple sclerosis.     

Astrocyte-derived TGF-beta 2 and NGF differentially regulate neural recognition molecule expression by cultured astrocytes

Because of the importance of neural recognition molecules expressed by glial cells to mediate interactions with neurons, growth factors and cytokines known to be functional during morphogenesis and in diseases of the nervous system were studied for their effects on recognition molecule expression by cultured immature and mature astrocytes from several brain regions. In cultures of immature astrocytes, transforming growth factors-beta 1 (TGF-beta 1) and -beta 2 (TGF-beta 2) and nerve growth factor (NGF) increased expression of the neural adhesion molecule L1, leading to a glia-mediated L1-specific increase in neurite outgrowth of dorsal root ganglion neurons on the astrocyte substrate. L1 expression induced by TGF-beta was inhibited by addition of antibodies to NGF, suggesting that TGF-beta influences L1 expression by modulating production of NGF by astrocytes. TGF-beta 1 and -beta 2 decreased expression of N-CAM by immature astrocytes. Since N-CAM expression was not affected by NGF and antibodies to NGF did not abolish the TGF-beta-induced decrease in N-CAM expression, NGF did not appear to be the mediator for regulating expression of N-CAM. Expression of the adhesion molecule on glia (AMOG) was not affected by any factor. NGF and TGF-beta 2 in latent form, but not TGF-beta 1 were found in the culture supernatants. Addition of interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF) to the cultures did not change recognition molecule expression. REcognition molecule expression by mature astrocytes was not found to be modified by any of the factors tested. In view of the observation that levels of L1 and N-CAM expression correlated with the presence of TGF-beta 2 and NGF in the culture supernatants of immature astrocytes, an autocrine regulatory mechanism for recognition molecule expression by these cells is suggested to play a crucial role in regulation of neuron-glia interactions.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide.

BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases preferentially expressed in macrophages and microglia and is active after prolonged incubation in neutral pH. Upon activation of macrophages by a number of inflammatory mediators, there is an increase in secreted cathepsin S activity accompanied by a decrease in cellular cathepsin S activity and protein content, as well as a decrease in cathepsin S mRNA. The decrease in cathepsin S mRNA and protein at the cellular level is in contrast to the response observed in some in vivo scenarios. MATERIALS AND METHODS: We investigated the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), two growth factors present during cell injury and inflammation but not known to activate macrophages and microglia, on the expression of cathepsin S, cathepsin B, and cathepsin L mRNAs in these cells, and on cathepsin S activity. We then tested the ability of cathepsin S to degrade myelin basic protein, and amyloid beta peptide at both acidic and neutral pH. RESULTS: Basic FGF and NGF treatment of macrophages and microglia significantly increased the levels of cathepsin S, B, and L mRNAs (2- to 5-fold). Basic FGF also increased cathepsin S activity intra- and extracellularly. Recombinant human cathepsin S was able to degrade myelin basic protein and monomeric and dimeric amyloid beta peptide at both acidic and neutral pH, as well as to process human amyloid precursor protein generating amyloidogenic fragments. CONCLUSIONS: These data suggest that bFGF and NGF may be the molecular signals that positively regulate the expression and activity of cysteine lysosomal proteases (cathepsin S in particular) in macrophages and microglia in vivo, and that there is an interplay between these factors and the activators of inflammation. Disruption of the balance between these two categories of signals may underlie the pathological changes that involve cysteine proteases. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p334. html     

Stimulatory effect of epidermal growth factor on the development of mouse early embryos in vitro.

Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo.     

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.     

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.     

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.     

Triptolide Upregulates NGF Synthesis in Rat Astrocyte Cultures

Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by d-amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson’s disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes. Authors Bing Xue and Jian Jiao contributed equally to this work.     

Interferons Suppress Nerve Growth Factor Synthesis as a Result of Interference with Cell Growth in Astrocytes Cultured from Neonatal Mouse Brain

Abstract: Interferon (IFN)-β and IFN-γ inhibited the DNA synthesis and nerve growth factor (NGF) synthesis in growing astrocytes cultured from neonatal mouse brain, but they did not affect the NGF synthesis in quiescent astrocytes. IFN-β and IFN-γ also inhibited the enhanced DNA synthesis and NGF synthesis in growing astrocytes after the administration of basic fibroblast growth factor. These results indicated that NGF synthesis in astrocytes is regulated by IFNs associated with cell growth. The mechanism of IFN action on NGF synthesis/secretion is unknown, but the results that their effects last long after IFN removal from the cultures present the possibility that IFNs destabilize NGF mRNA.     

Induction of NGF synthesis in astrocytes by onjisaponins of Polygala tenuifolia, constituents of kampo (Japanese herbal) medicine, Ninjin-yoei-to.

The effect of a kampo medicine, Ninjin-yoei-to (NYT; Ren-shen-yang-rong-tang in Chinese) on nerve growth factor (NGF) secretion from the cultured rat astrocytes was examined in vitro. When rat embryo astrocytes were cultured in the presence of NYT for 24 h, the amount of NGF in the medium was significantly increased in a dose dependent manner. Among 14 kinds of component herbs in NYT, the roots of Polygala tenuifolia and roots of Panax ginseng extracts increased NGF levels from the astrocytes. Saponin fraction from the roots of P. tenuifolia enhanced the production of NGF, however phenolic glycoside fraction showed no effect. Onjisaponins A, B, E, F and G as major saponins of the root of P. tenuifolia strongly increased the NGF level, whereas ginsenosides Rb1 and Rg1 did not affect the NGF level. Onjisaponin F also induced ChAT mRNA level in rat basal forebrain cells. These results indicate the possibility that NYT and/or onjisaponins in P. tenuifolia may have potential therapeutic effects for the treatment of Alzheimer disease patients.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

Regulation by Interleukin-1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.     

高压氧治疗创伤性脑损伤的效用及机制研究

目的:研究高压氧(HBO)对大鼠创伤性脑损伤(TBI)治疗效用并观察脑组织星形胶质细胞活化及胶质细胞源性神经营养因子(GDNF)和神经生长因子(NGF)表达的变化以探讨作用机制。方法:SD雄性大鼠54只,随机分为3组(n=18):假手术组、TBI组和HBO治疗组。采用Feeney法建立大鼠TBI模型,假手术组只开放骨窗,不予打击。HBO治疗组大鼠于脑损伤后6 h采用动物高压舱,以3ATA压力纯氧治疗60 min。TBI后48 h测量神经功能,然后分离脑组织,其中18只用干湿法测定脑含水量;18只脑组织用于切片,部分进行尼氏染色后作形态学观察,部分进行免疫组织化学染色,检测星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)、波形蛋白(vimentin)与S100蛋白的表达;另18只大鼠取伤侧脑半球,进行Western blot分析,观察GDNF和NGF的表达。结果:HBO治疗能减轻神经功能障碍,降低脑含水量,减少海马部位神经细胞丢失,进一步激活损伤侧皮质与海马部位GFAP、vimentin与S-100阳性表达星形胶质细胞,促进损伤侧脑组织GDNF与NGF的表达。结论:HBO对创伤性脑损伤有较好治疗效果,其机制与上调GDNF和NGF的表达有关。     

Differential regulation and function of Fas expression on glial cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.