Levels of transforming growth factor alpha (TGF-a)in human cerebrospinal fluid

In this study, we investigated cerebrospinal fluid of patients with various neurologicalsymptoms for the presence of transforming growth factor alpha (TGF-a). 41 samples ofcerebrospinal fluid were collected by lumbar puncture performed routinely due to the clinicalsuspicion of neurological disease from 22 females (age 15–80 years, median 42 years) and from19 males (age 18–82 years, median 48 years). A highly sensitive and specific radioimmunoassaywas used to determine the concentration of TGF-a in the samples. The detection limit of the assaywas about 200 pg TGF-a. There was no cross-reactivity to human EGF. We showed CSF indeeddoes contain TGFa. As TGF-a was detected in all 41 samples investigated, this growth factorappears to be a constant component of CSF. The mean concentration was 5.5 ng TGF-a (S.D.±2.7 pg/ml, range 1.1 to 13.9 pg/ml). There was no significant correlation between TGF-aconcentration in CSF and age (r=−0.006) and there was no significant differencebetween females (mean 5.8±3.10 pg/ml) and males (mean 5.2±1.96 pg/ml). No diagnosis wasover represented in patients with TGF-a concentrations above or below 1 S. D. off the mean.However, highest concentrations of TGF-a were found in the group of patients with peripheralneurological sensory dysfunctions and polyneuropathy. We conclude that TGF-a is not only aconstant component of human cerebrospinal fluid in adults but could also be significantly involvedin the pathophysiology of various neurological diseases. The earlier hypothesis that TGF-a couldmainly have a role in brain development needs hence to be re-evaluated.     

Multiple trophic actions of heparin-binding epidermal growth factor (HB-EGF) in the central nervous system

The epidermal growth factor (EGF) family of ligands interacts with the epidermal growth factor receptor (EGF-R) to produce numerous direct and indirect actions on central nervous system cells. They induce the proliferation of astrocytes and multipotent progenitors ('stem' cells) and promote the survival and differentiation of postmitotic neurons. Heparin-binding epidermal growth factor (HB-EGF) interacts with both EGF-R and a related receptor, ErbB4, whereas transforming growth factor alpha (TGFalpha) interacts only with EGF-R. Because of the unique characteristics of HB-EGF and the potential utility of EGF family members in brain repair, we examine the effects of HB-EGF on rat and mouse CNS cells in vitro and compare them to those of TGFalpha. We find that, like TGFalpha, HB-EGF stimulates the proliferation of CNS astrocytes and multipotent progenitors. These proliferative effects require the expression of EGF-R, as no such effects are observed in cells derived from EGF-R-/- mice. Both HB-EGF and TGFalpha enhanced the survival of neurons derived from the neocortex and the striatum. Within these neuron-enriched cultures, nestin-positive cells but not neurons express EGF-R mRNA, indicating that the neurotrophic actions of EGF-R ligands are a result of indirect stimulation mediated by non-neuronal cells. The neurotrophic actions of HB-EGF and TGFalpha are accompanied by an elevation in immunoreactive dual phosphorylated mitogen-activated protein kinase (MAP kinase) in neurons, providing evidence that the MAP kinase cascade mediates these actions. In situ hybridization studies demonstrate that HB-EGF mRNA is present within the brainstem as early as E14 and subsequently is found in the developing cortical plate, hippocampus, cerebellar Purkinje cells and ventrobasal thalamus, among other brain areas. These findings indicate that HB-EGF may be an important trophic factor in the developing CNS and is a useful candidate molecule for brain repair strategies.     

Postsynaptic expression of an epidermal growth factor receptor regulates cholinergic synapse formation between identified molluscan neurons

Epidermal growth factor (EGF) family members are conserved in both vertebrates and invertebrates. Recent studies suggest that EGF ligands in invertebrates may have neurotrophic actions that possibly compensate for the apparent absence of neurotrophins in these species. In this study, we have cloned an EGF receptor from the mollusk Lymnaea stagnalis (L-EGFR), and shown that L-EGFR is the receptor for a previously identified EGF-like peptide in Lymnaea , named Lymnaea EGF (L-EGF). Knock-down of L-EGFR expression prevented L-EGF-induced excitatory synapse formation between identified cholinergic neuron visceral dorsal 4 (VD4) and its postsynaptic partner left pedal dorsal 1 (LPeD1). Moreover, knock-down of L-EGFR also prevented synapse formation induced by Lymnaea brain conditioned medium, suggesting that L-EGF is the most important, if not the only, brain-derived factor that promotes excitatory cholinergic synapse formation in Lymnaea . Thus, our data establish canonical EGF/EGFR signaling as an important synaptotrophic mechanism in invertebrates.     

Antisense epidermal growth factor receptor RNA transfection in human malignant glioma cells leads to inhibition of proliferation and induction of differentiation

The epidermal growth factor receptor (EGFR) is a proto-oncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased ( P <0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G₀ /G₁ phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.     

F90对胶质母细胞瘤细胞的作用及机制研究

目的探讨表皮生长因子受体（EGFR）抑制剂F90对胶质母细胞瘤（GBM）细胞的抑制作用及其机制。方法采用MTT法检测F90对．GBM细胞的抑制作用，流式细胞术检测细胞凋亡和免疫印迹法（Western Blot）检测蛋白表达。结果F90能明显抑制U251和SHG-44细胞的生长，半数抑制浓度（IC50）分别为（5．8±113）和（5．1±1．2）μmol／L，且呈一定的剂量相关性。F90可以诱导U251细胞的凋亡，抑制U251细胞EGFR的磷酸化及其丝裂原活化的蛋白激酶（MAPK）通路下游信号的活化，下调Bcl-2蛋白的表达，上调P53蛋白的表达。结论F90能有效抑制某些GBM细胞在体外的生长，与Iressa作用强度相当，有可能成为一种新的治疗GBM的药物。     

FGF和EGF对神经干细胞增殖及分化的影响

胚胎和成年哺乳动物脑内均存在的神经干细胞,成纤维细胞生长因子和表皮生长因子对神经干细胞的增殖及分化有一定的影响,ＦＧＦ和ＥＧＦ及其受体在胚胎期和成年期表达各异。ＦＧＦ和ＥＧＦ能促进神经干细胞增殖,在不同的条件下对分化和作用不同。     

EGF-induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons

Epidermal growth factor (EGF), over a low range of concentrations (165-825 pM), induced neuritogenesis in post-mitotic chick CNS precursor neurons cultured in a serum-free medium, without the addition of other growth factors. Antibody to EGF blocks the neurite-promoting activity of EGF. Similarly, neuritogenesis of cultured chick CNS neurons in medium supplemented with 20% fetal bovine serum is blocked by antibody to EGF, even though serum may contain other neuronotrophic bioactive proteins and steroids. Quantitatively, the only major gangliosides of the undifferentiated post-mitotic neurons are GD3 and GD2. GD3 as well as its biosynthetic precursor, GM3, undergo active biosynthesis in serum-free medium as evidenced by their vigorous labeling by radioactive galactose supplied in the culture medium. When the undifferentiated neurons in serum-free medium are exposed to EGF, the ensuing generation of neurite plasma membrane coincides with initiation of biosynthesis of the sialosyl gangliotetraosyl ceramide species of gangliosides (GD1A, GD1B, GT1B, GQ1B). Antibody to EGF simultaneously inhibits biosynthesis of these gangliosides as well as inhibition of neuritogenesis. These findings indicate that EGF may be a primary neurite-inducing growth factor for post-mitotic embryonic CNS neurons and that gangliosides, particularly those of the sialosyl gangliotetraosyl ceramide species, characterize the plasma membrane of CNS neurons during neuritogenesis.     

Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Reduction of nerve growth factor receptor immunoreactivity in ischaemic gerbil hippocampal CA1 neurons after treatment with L-threo-3,4-dihydroxyphenylserine (DOPS)

Abstract

Nerve growth factor (NGF) synthesis in cultured mouse L-M fibroblast and astroglial cells can be increased after the treatment with L-threo-3,4-dihydroxyphenylserine (DOPS). Since the increase of NGF is not blocked by the treatment with decarboxylase inhibitor, DOPS may have direct effect to increase the NGF content. NGF and its receptor (NGFR) are suggested to play an important role in the neuronal survival and regeneration under pathologic conditions. In this study, we studied a possible protective effect of DOPS against the hippocampal CA1 cell death after transient forebrain ischaemia in gerbils in relation to the change of NGFR immunoreactivity. We found that treatment with DOPS (300 mg kg^–1) in combination with a decarboxylase inhibitor (benserazide, 10 mg kg^–1) protected ischaemic hippocampal CA1 cell against delayed neuronal death (neuronal density = 125 ± 24 mm^–1) as compared to the treatment with vehicle (49 ± 11 mm^–1) (p < 0.01). The immunoreactivity for NGFR was scarcely present in the sham-control CA1 area but was induced from 1 h and markedly expressed at 7 days after recirculation in the vehicle group. However; it was slightly and transiently induced from 8 h to 2 days in the DOPS plus benserazide treated group. These data suggest that the protective role of DOPS on the ischaemic hippocampal CA1 cells may act through the NGF and its receptor system. [Neurol Res 1994; 16: 201–204]     

EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture.

Epidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia. In this study, we investigated the effect of EGF on cells cultured from 16-day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo. EGF receptors were detected in untreated cultures by [125I]-EGF binding. Treatment of the cultures with EGF resulted in up to 50-fold increases in neuronal high-affinity dopamine uptake. Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase-immunoreactive cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons. By contrast, the high-affinity uptake for serotonin was increased only threefold over its controls. There was no significant effect on high-affinity gamma-aminobutyric acid (GABA) uptake. These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures. Immunocytochemistry for glial fibrillary acidic protein demonstrated an increase in astroglia with EGF treatment. Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.     

Bone morphogenetic protein 4, inhibitor of differentiation 1, and epidermal growth factor receptor regulate the survival of cochlear sensory epithelial cells

Bone morphogenetic protein 4 (BMP4) is crucial for the development of the inner ear, but the mechanism of how BMP4 affects this process remains unknown. The focus of this research was to determine whether BMP4 can regulate the survival of cochlear sensory epithelial cells through inhibitor of differentiation 1 (Id1) and the epidermal growth factor receptor (EGFR). In this study, we first investigated the effects of BMP4, noggin, and dominant negative BMPR1B (dnBMPR1B) on the proliferation and survival of cochlear sensory epithelial cells. Subsequently, we investigated the influences of BMP4, noggin, and dnBMPR1B on the expression of Id1 and EGFR. We found that BMP4 had a negative effect on cell survival and on Id1 and EGFR expression in vitro, whereas noggin and dnBMPR1B treatment had positive effects. Knockdown of the Id1 gene with short interfering RNA (siRNA) reduced the expression of EGFR and cell proliferation. These data suggest that BMP4 may regulate survival through the Id1/EGFR pathway during development of the rat inner ear. © 2013 Wiley Periodicals, Inc.     

Effects of cotransfection of antisense‐EGFR and wild‐type PTEN cDNA on human glioblastoma cells

The main molecular genetic changes identified in glioblastomas are overexpression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/deletion of the tumor suppressor PTEN gene. These two genetic changes both play important roles in glial tumorigenesis and progression. In this study, we demonstrated that wild‐type PTEN transfection inhibited the growth and transforming ability of U87MG cells by 69.3% and 73.5%, respectively. On the other hand, antisense‐EGFR transfection inhibited the growth and transforming phenotype of these cells by 50.3% and 46.8%, respectively. However, cotransfection of U87MG cells with wild‐type PTEN and antisense EGFR constructs could inhibit the cellular growth by 91.7%. The transforming phenotype of these cells was completely inhibited. In addition, these cotransfected cells showed a differentiated form and expressed much lower telomerase activity than cells transfected with wild‐type PTEN or antisense‐EGFR alone. In summary, these results suggest that cotransfection is a better approach to suppress glioma cell growth than wild‐type PTEN transfer or antisense‐EGFR transfection alone. This approach may prove useful as an adjunct therapy in the treatment of glioblastomas.     

Cytoarchitectonics of substantia nigra grafts: a light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes

Maturation of dopaminergic (DA) neurons and astroglia was studied in transplants of the substantia nigra grown for up to 7 months in the brain of rats. The investigation had three specific aims. The first was to observe effects of different transplant positions on the longevity of DA neurons. Second, the grafts were examined for changes of synaptic interactions and associations between DA neurons and astroglia. Third, an answer was sought to the question whether transplanted DA neurons migrate into the adjacent host brain. The grafts were taken from the ventral mesencephalon of rat embryos of different ages (day 14 to 18 of gestation) and placed into the cerebral cortex, tectum, cerebellum, or ventricles of newborn host animals. Following different times of survival the immunocytochemical localization of tyrosine hydroxylase (TH) and of glia filament protein (GFA) in the transplants were observed. In all of the transplantation sites, except for one, neurons of different morphologies that contained TH were found in the grafts. The cerebellar white matter of the host brain failed to support the long-term survival of DA neurons. The overall structure of mature substantia nigra grafts had some resemblance to intact substantia nigra (SN). On the ultrastructural level, it was found that morphological expression of some immature features of DA neurons, such as glial sheaths, somatic spines, and lack of oligodendroglia, persisted in mature grafts. Specific associations of DA neurons and astroglia in the grafts suggested that the cytoarchitectonic appearance of a given brain region may be related to the existence of particular neuron glia relationships. In contrast to intact SN, transplants revealed deficiencies in unlabeled pleomorphic boutons and contained some TH-immunoreactive terminals. Migration of DA neurons and their processes into the adjacent host brain was rarely observed.     

碱性成纤维细胞生长因子、表皮生长因子对脑缺血大鼠内源性神经干细胞增殖的影响

目的探讨碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对大鼠局灶性脑缺血模型内源性神经干细胞增殖的影响。方法通过大脑中动脉阻塞法建立大鼠的局灶性脑缺血模型,随机分组后分别皮下注射生理盐水、bFGF、EGF以及bFGF EGF;每日1次,共3d,此后每3d1次。采用免疫组化法,以5溴脱氧嘧啶尿苷(Brdu)标记神经干细胞,观察并比较各组大鼠制模后第7d、14d、21d侧脑室室管膜下区(SVZ)和海马齿状回Brdu阳性细胞的表达。结果制模后,各组大鼠双侧SVZ和海马齿状回均出现Brdu阳性细胞,且阳性细胞数随时间递减;与对照组相比,药物干预组Brdu阳性细胞数显著增加(P<0.05～0.01);与单药组相比,bFGF EGF组(联合组)Brdu阳性细胞数增加更明显(P<0.05～0.01);各药物干预组在制模第7dBrdu阳性细胞数最多(P<0.05～0.01)。结论皮下注射bFGF、EGF可促进脑缺血大鼠模型内源性神经干细胞的增殖;bFGF和EGF联合应用对脑缺血大鼠神经干细胞的增殖效应有协同作用。     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用

目的观察神经生长因子（nerve growth factor, NGF）对原代培养的背根神经节（dorsal root ganglion, DRG）神经元中P物质（substance P, SP）的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体（vanilloid receptor 1, VR1）mRNA的表达,用放射免疫分析（radioimmunoassay,RIA）法检测SP的基础释放量和辣椒素（100 nmol/L）刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用(英文)

目的观察神经生长因子(nerve growth factor, NGF)对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中P物质(substance P, SP)的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体(vanilloid receptor 1, VR1)mRNA的表达,用放射免疫分析(radioimmunoassay,RIA)法检测SP的基础释放量和辣椒素(100 nmol/L)刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

Norepinephrine activates extracellular-regulated kinase in cortical neurons.

BACKGROUND: Previous studies demonstrate that indirect activation of monoamine receptors by antidepressant treatment increases neurotrophic factors that activate the mitogen-activated protein kinase cascade; however, it is also possible that these monoamine receptors influence the mitogen-activated protein kinase pathway independent of neurotrophic factors. The influence of norepinephrine on the phosphorylation of extracellular-regulated protein kinase is characterized. METHODS: Primary cerebral cortical cultures were prepared from embryonic day 18 rat brains and were subsequently incubated with norepinephrine in the absence or presence of agents acting as noradrenergic receptors or as intracellular signaling proteins. Levels of phosphorylated extracellular-regulated protein kinase were determined by immunoblot. RESULTS: The results demonstrate that incubation with norepinephrine produces a time- and dose-dependent activation of phosphorylated extracellular-regulated protein kinase and that this increase is dependent on activation of alpha(2)- and beta-adrenergic receptor subtypes. In addition, the results demonstrate that norepinephrine activation of phosphorylated extracellular-regulated protein kinase is dependent on a pertussis toxin-sensitive G protein, a receptor tyrosine kinase, and activation of phosphatidylinositol 3-kinase. CONCLUSIONS: The findings suggest that activation of the mitogen-activated protein kinase cascade by norepinephrine can occur via a tyrosine kinase-dependent signaling pathway but independent of classical second-messenger or Src-dependent kinases.     

Prognostic significance of Ki67, p53 and epidermal growth factor receptor immunostaining in human glioblastomas

gabctgPrognostic significance of Ki67, p53 and epidermal growth factor receptor immunostaining in human glioblastomas
Since glioblastomas in adults are uniformly fatal, evaluation of easily reproducible prognostic criteria which would attempt to define groups of patients is required. However, there is lack of a clear consensus regarding the expression of some markers in the literature. Therefore, an immunohistochemical study was performed to determine the prognostic significance of Ki67, p53, and epidermal growth factor receptor (EGFR) in a retrospective series of 63 glioblastomas. Image analysis was carried out in positive specimens to quantify the immunoprecipitates. p53 and EGFR expression were specifically addressed in the 36 primary glioblastomas reported in this series. In all cases, clinical data (age, Karnofsky performance scale index [KPS] before surgery, extent of surgery) and immunohistochemical features were analysed using univariate and multivariate analysis to ascertain whether any significant correlation exists between [1] EGFR expression [2], p53 accumulation [3], Ki67 labelling index and prognosis (survival time and disease-free survival time, DFST). The results showed that in this series of glioblastomas, none of these markers had any prognostic value. Among the clinical parameters, a high KPS before surgery was found to be indicative of a shorter DFST and survival time ( P <0.05), whereas a younger age at onset and total or subtotal surgical excision were associated with a longer survival ( P <0.001 and 0.05, respectively). EGFR protein accumulation was inversely correlated with p53 accumulation ( P =0.01). The percentage of the primary glioblastomas expressing EGFR was much lower in our study (33%) than in the literature suggesting that the molecular distinction between primary and secondary glioblastomas is not so clear-cut.