Neurotrophic effect of basic fibroblast growth factor is mediated by the p42/p44 mitogen-activated protein kinase cascade in cultured rat cortical neurons

Basic fibroblast growth factor (bFGF) has been reported to support the survival of brain neurons. In the present study, we investigated whether the neurotrophic effect of bFGF is mediated by the mitogen-activated protein kinase (MAPK) cascade in cultured rat cerebral cortical neurons. Recombinant human bFGF (0.1-10 ng/ml) induced phosphorylation of p44/42 MAPK (ERK1/2) in a concentration- and time-dependent manner. bFGF-induced ERK1/2 phosphorylation and promotion of neuronal survival were both blocked by U0126 and PD98059, inhibitors of the MAPK-activating enzyme MEK. These results suggest that the MEK/ERK signal transduction cascade is involved in the neurotrophic effect of bFGF.     

Basic fibroblast growth factor induces TGF-beta release in an isoform and glioma-specific manner

The aim of the current study was to determine whether basic fibroblast growth factor (bFGF), regulates the release of transforming growth factor-beta1 (TGF-beta1) from C6 glioma cells. The results of the study show that bFGF (2, 5 and 10 ng/ml) dose dependently induced the release of TGF-beta1 from C6 glioma cells, with the 10 ng/ml dose inducing a 2- to 3-fold increase of TGF-beta1 levels. This effect was evident as early as 6 h following treatment, with maximal levels observed at 18 h. The effect of bFGF was largely on latent TGF-beta1, and was isoform specific, as bFGF had no effect on TGF-beta2 release. The bFGF effect on TGF-beta1 was also glioma specific, as no such stimulatory effect was observed in rat cortical astrocytes.     

Following nerve injury neuregulin-1 drives microglial proliferation and neuropathic pain via the MEK/ERK pathway

Following peripheral nerve injury microglia accumulate within the spinal cord and adopt a proinflammatory phenotype a process which contributes to the development of neuropathic pain. We have recently shown that neuregulin-1, a growth factor released following nerve injury, activates erbB 2, 3, and 4 receptors on microglia and stimulates proliferation, survival and chemotaxis of these cells. Here we studied the intracellular signaling pathways downstream of neuregulin-1-erbB activation in microglial cells. We found that neuregulin-1 in vitro induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. Using specific kinase inhibitors we found that the mitogenic effect of neuregulin-1 on microglia was dependant on MEK/ERK1/2 pathway, the chemotactic effect was dependant on PI3K/Akt signaling and survival was dependant on both pathways. Intrathecal treatment with neuregulin-1 was associated with microgliosis and development of mechanical and cold pain related hypersensitivity which was dependant on ERK1/2 phosphorylation in microglia. Spinal nerve ligation results in a robust microgliosis and sustained ERK1/2 phosphorylation within these cells. This pathway is downstream of neuregulin-1/erbB signaling since its blockade resulted in a significant reduction in microglial ERK1/2 phosphorylation. Inhibition of the MEK/ERK1/2 pathway resulted in decreased spinal microgliosis and in reduced mechanical and cold hypersensitivity after peripheral nerve damage. We conclude that neuregulin-1 released after nerve injury activates microglial erbB receptors which consequently stimulates the MEK/ERK1/2 pathway that drives microglial proliferation and contributes to the development of neuropathic pain.     

Biomodulatory role of ceramide in basic fibroblast growth factor-induced proliferation of cerebellar astrocytes in primary culture

To evaluate the role of ceramide in glial growth, primary cultures of quiescent astrocytes from rat cerebellum were stimulated to proliferate by mitogenic doses of basic fibroblast growth factor (bFGF). Parallel to the bFGF mitogenic effect was a marked, and persistent, decrease in cellular ceramide levels. Both in vitro and in culture metabolic studies have led us to exclude both sphingomyelinase and ceramidase involvement in ceramide level variation. Instead, we found evidence of a functional connection between the decrease in ceramide levels and astrocyte proliferation. In fact, cell growth in bFGF-stimulated astrocytes was inhibited by exogenous ceramide and C2-ceramide, maximal inhibition being obtained at a ceramide concentration of 5-10 microM. Under the same conditions, the dihydroderivatives of ceramides were without effect. Following ceramide treatment, the phosphorylation of the MAP kinase isoforms ERK1/2, key components in bFGF-induced cell proliferation, was examined. The administration of antiproliferative doses of ceramide or C2-ceramide, but not of their dihydroderivatives, resulted in a significant inhibition of ERK1/2 activation. In conclusion, our data indicate that the prompt modulation of ceramide levels by bFGF is an early step associated with the signaling pathways responsible for the mitogenic activity of bFGF in astrocytes.     

Astrocyte growth effects of vascular endothelial growth factor (VEGF) application to perinatal neocortical explants: receptor mediation and signal transduction pathways

The non-angiogenic role of vascular endothelial growth factor (VEGF), and its receptors flt-1 and flk-1, together with downstream signaling pathways were examined in fetal and postnatal rat cerebral cortical organotypic explants. VEGF application in both paradigms caused a significant increase in astroglial proliferation and a dose-dependent increase in GFAP and nestin immunoreactivity. The VEGF receptor flt-1 was observed on most, though not all astrocytes, while flk-1 receptor immunoexpression was absent. Treatment with antisense oligonucleotides (AS-ODNs) to flt-1 resulted in a dramatic decrease in GFAP and nestin immunoreactivity, which further confirmed the role of flt-1 in mediating VEGF's gliotrophic effects, while AS-ODNs to flk-1 had no effect. VEGF-induced gliotrophic effects were found to be mediated by the MAPK/ERK and PI-3 kinase signaling pathways, since the both the MEK1 inhibitor, PD98059 and the PI-3 kinase inhibitor, Wortmannin abolished VEGF-induced astrocytic GFAP(+) expression. Although high dose VEGF application resulted in strong upregulation of both GFAP and nestin immunoreactivity in astrocytes, overlap of the two proteins was not observed in all cells, suggesting that some of the nestin(+) cells might be neural progenitors. Exposure to VEGF resulted in upregulation of both VEGF and bFGF mRNA at the one-day time point, and bFGF protein by 3 days; VEGF activated astrocytes expressed bFGF to a much greater degree than those in untreated explants. The increased expression of bFGF induced by VEGF, may serve in the proliferation of multipotential neural stem/progenitor cells in vitro. VEGF, an established angiogenic factor, appears to play a significant role in the growth and differentiation of astrocytes in the CNS.     

Activation of adenosine A1 receptor-induced neural stem cell proliferation via MEK/ERK and Akt signaling pathways

Adenosine, a modulator of neuronal function in the mammalian central nervous system, exerts a neuroprotective effect via the adenosine A(1) receptor; however, its effect on neural stem cells (NSCs) remains unclear. Because adenosine is released in response to pathological conditions and NSCs play a key role in neuroregeneration, we tested the hypothesis that adenosine is capable of stimulating NSC proliferation. We demonstrated that NSCs dominantly express adenosine A(1) and A(2B) receptors. Adenosine and the adenosine A(1) receptor agonist cyclopentyladenosine (CPA) increased proliferation of NSCs, and this CPA-induced cell proliferation was attenuated by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). CPA also induced phosphorylation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase/ERK kinase (MEK), and Akt, and their phosphorylation was inhibited by DPCPA. In addition, CPA-induced cell proliferation was inhibited by MEK and Akt inhibitors. These results suggest that activation of adenosine A(1) receptor-stimulated proliferation of NSCs occurs via MEK/ERK and Akt signaling pathways.     

Intersectin1‐s,A multidomain adapter protein,Is essential for malignant glioma proliferation

Glioblastomas, the most aggressive form of primary brain tumors with a tendency to invade surrounding healthy brain tissues, remains an incurable disease. Intersectin (ITSN) is a multidomain adapter protein implicated in endocytosis, exocytosis, and multiple signaling pathways. Prior research of ours has shown intersectin1‐S (ITSN1‐S) is critical for the migration and invasion of glioma cells by regulating several key proteins. In this study, we established ITSN1‐S expression patterns in human tumor tissues. We discovered that ITSN1‐S expression was positively correlated with histological grade of gliomas and with poor patient prognosis. We also found that the expression of ITSN1‐S protein was essential to glioblastoma cell proliferation. Furthermore, through a series of expression constructs encoding different ITSN1‐S domains, we identified the critical roles of ITSN1‐S SH3 domains in the regulation of cell proliferation. This study also demonstrates evidence suggesting that the regulation of ITSN1‐S on glioblastoma cells proliferation is through the Raf/MEK/ERK pathway. In conclusion, this study suggests critical roles of ITSN1‐S in malignant glioma proliferation, indicating a potential usage of ITSN1‐S in the therapeutic intervention as a novel molecular target. GLIA 2015;63:1595–1605     

Caveolin-1 inhibits neurite growth by blocking Rac1/Cdc42 and p21-activated kinase 1 interactions

Growth factors such as basic fibroblast growth factors (bFGFs) could induce the differentiation of mouse neuroblastoma cells. We examine the effect of caveolin-1 on bFGF-induced differentiation of N2a cells. Caveolin-1 blocked the formation of neurites and the phosphorylation of Erk upon bFGF treatment in N2a cells. Active mutants of Rho family small GTPases (Rac1 and Cdc42) could not affect the inhibitory effect of caveolin-1, but we could restore the differentiation of N2a cells by introducing active mutants of p21-activated kinase 1 (PAK1). Over-expressed caveolin-1 could be coimmunoprecipitated with PAK1, which interrupted the steady-state Rac1/Cdc42-PAK1 interactions. From these results, we suggest that the up-regulated caveolin-1 in neuronal cells can inhibit the bFGF signaling pathway from small GTPases to PAK1 by directly binding to PAK1.     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节(英文)

目的静脉注射碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK)-细胞外信号调节激酶(extracellular signal-regulated kinase, ERK) 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1(early growth respons factor 1, Egr-1)的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

MAPK/ERK激酶-ERK信号通路参与外源性bFGF对缺氧-复氧损伤后星形胶质细胞内Egr-1结合活性的调节

目的 静脉注射碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）可以明显降低实验性脑缺血大鼠的脑梗死面积,但该作用的分子机制尚不清楚。本文旨在研究外源性bFGF 作用的信号转导通路。方法缺氧-复氧损伤星形胶质细胞。Western blot检测外源性bFGF作用后丝裂原活化蛋白激酶/细胞外信号调节激酶激酶（mitogen-activated protein kinase/extracellular signal-regulated kinase kinase,MEK）-细胞外信号调节激酶（extracellular signal-regulated kinase, ERK） 信号通路活化。电泳变动迁移率分析实验检测外源性bFGF 作用后核转录因子早期生长反应因子-1（early growth respons factor 1, Egr-1）的结合活性变化。结果外源性bFGF可以保护胞外信号调节激酶MEK-ERK信号通路蛋白不被氧自由基降解。MEK-ERK信号通路在外源性bFGF作用后活化。这一信号通路进一步使Egr-1结合活性升高。结论外源性bFGF可能通过激活ERK信号通路,促进内源性转录因子Egr-1的结合活性升高,进而促进内源性bFGF的表达。     

Sphingosine-1-phosphate is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation through Gi-protein-coupled receptors

The mitogenic role of sphingosine-1-phosphate (S1P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S1P formation, bFGF inducing astrocytes to release S1P, but not sphingosine kinase, in the extracellular milieu. The SK inhibitor N,N-dimethylsphingosine inhibited S1P release as well as bFGF-induced growth stimulation. S1P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S1P, mimicked by the S1P receptor agonist dihydro-S1P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S1P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S1P but did not affect DNA synthesis. These results support the view that S1P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S1P receptors. In agreement, mRNAs for S1P1, S1P2, and S1P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:1204-1215). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum 1:129-135), efficiently inhibited S1P-induced proliferation. The S1P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S1P and bFGF/S1P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.     

Basic fibroblast growth factor (bFGF) and rat C6 glioma cells: regulation of expression, absence of release, and response to exogenous bFGF

Basic fibroblast growth factor (bFGF) is a potent mitogen for several types of cells, including glial cells, which also seem to express bFGF. We have used rat C6 glioma cells as a model system to study the expression and release of bFGF by glioma cells, as well as the effects of exogenous bFGF on these cells. We have shown that C6 cells express 18 kD bFGF and several higher molecular weight immunoreactive forms. The expression of bFGF could be induced by a factor present in fetal calf serum. Subsequent to its initial appearance, bFGF is regulated in a cell density-dependent manner. Neither bFGF-like immunoreactive material, nor bFGF-like neurotrophic activity were found to be released by C6 cells. Exogenously applied bFGF changed C6 cell morphology similar to cyclic AMP induced alterations but had no significant influence on C6 cell proliferation and biochemical differentiation. From these results we conclude that bFGF in C6 cells might act as an endogenous (not autocrine) mitogen. Possible roles for bFGF in glial cells are discussed.     

抑制SSRP1对胶质瘤细胞增殖、化疗敏感性的影响

目的 探讨结构特异性识别蛋白1(SSRP1)与胶质瘤预后的相关性,以及抑制SSRP1对胶质瘤细胞增殖及化疗敏感性的影响。方法 应用生信分析方法,检索中国胶质瘤数据库CGGA数据集,分析SSRP1表达与胶质瘤预后相关性。体外培养胶质瘤U251、U87细胞,应用CBL0137抑制SSRP1,应用替莫唑胺（TMZ）检测化疗敏感性。应用CCK8法检测细胞增殖,利用免疫印迹法检测MAPK信号通路（p-38、ERK及JNK）表达。结果 生信分析结果显示,胶质瘤SSRP1呈高表达,随胶质瘤级别增加,SSRP1表达明星上调（P<0.05）;SSRP1高表达胶质瘤总生存期明显缩短（P<0.05）。抑制SSRP1,明显抑制体外培养U251、U87细胞增殖（P<0.05）,增加U251、U87细胞对TMZ化疗敏感性（P<0.05）,对U251、U87细胞p-38、ERK及JNK蛋白表达水平无明显影响,但明显降低U251、U87细胞p-38、ERK及JNK蛋白磷酸化水平（P<0.05）。结论 胶质瘤SSRP1呈高表达,与病人不良预后有关;抑制SSRP1,明显抑制胶质瘤细胞增殖,并...     

Tissue kallikrein protects rat hippocampal CA1 neurons against cerebral ischemia/reperfusion‐induced injury through the B2R‐Raf‐MEK1/2‐ERK1/2 pathway

We have documented that tissue kallikrein (TK) prevents neurons from hypoxia/reoxygenation injury through the B2R‐ERK1/2 pathway and the antihypoxic function of TK through Homer1b/c‐ERK1/2 signaling pathways. The present study investigates the molecular mechanisms of exogenous TK activation of the B2R‐ERK1/2 pathway through the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module in vivo. The cresyl violet staining results indicated that exogenous TK protected the rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. The immunoprecipitation (IP) and immunoblotting (IB) results revealed that exogenous TK upregulated the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and upregulated the phosphorylation of Raf (p‐Raf), MEK1/2 (p‐MEK1/2), and ERK1/2 (p‐ERK1/2). Meanwhile, exogenous TK upregulated the expression of nuclear factor‐κB (NF‐κB), depressed the release of cytochrome c (Cyt c) and bax from mitochondria to the cytosol, and depressed the activation of caspase‐3. Take together, our results suggest that exogenous TK attenuated the cerebral I/R induced rat hippocampal CA1 neurons injury through activating the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and that the activated B2R‐Raf‐MEK1/2 signaling module could upregulate the expression of NF‐κB, decrease the release of cytochrome c and bax from mitochondria to the cytosol, and depress the activation of caspase‐3. © 2014 Wiley Periodicals, Inc.     

Heparin-binding epidermal growth factor-like growth factor stimulates cell proliferation in cerebral cortical cultures through phosphatidylinositol 3'-kinase and mitogen-activated protein kinase

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.     

Role of ERK map kinase and CRM1 in IL‐1β‐stimulated release of HMGB1 from cortical astrocytes

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high‐mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL‐1β for 24 h elicited a dose‐dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL‐1β induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho‐ERK were increased by IL‐1β, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL‐1β increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL‐1β‐stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL‐1β stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke. © 2010 Wiley‐Liss, Inc.     

Basic Fibroblast Growth Factor (bFGF) Facilitates Differentiation of Adult Dorsal Root Ganglia-Derived Neural Stem Cells Toward Schwann Cells by Binding to FGFR-1 Through MAPK/ERK Activation

Considerable research has been devoted to unraveling the regulation of neural stem cell (NSC) differentiation. The responses of NSCs to various differentiation-inducing stimuli, however, are still difficult to estimate. In this study, we aimed to search for a potent growth factor that was able to effectively induce differentiation of NSCs toward Schwann cells. NSCs were isolated from dorsal root ganglia (DRGs) of adult rats and identified by immunostaining. Three different growth factors were used to stimulate the differentiation of DRG-derived NSCs (DRG-NSCs). We found that among these three growth factors, bFGF was the strongest inducer for the glial differentiation of DRG-NSCs, and bFGF induced the generation of an increased number of Schwann cell-like cells as compared to nerve growth factor (NGF) and neuregulin1-β (NRG). These Schwann cell-like cells demonstrated the same characteristics as those of primary Schwann cells. Furthermore, we noted that bFGF-induced differentiation of DRG-NSCs toward Schwann cells might be mediated by binding to fibroblast growth factor receptor-1 (FGFR-1) through activation of MAPK/ERK signal pathway.     

Acute, chronic and withdrawal effects of the cannabinoid receptor agonist WIN55212-2 on the sequential activation of MAPK/Raf-MEK-ERK signaling in the rat cerebral frontal cortex: short-term regulation by intrinsic and extrinsic pathways

The cannabinoids (CB) modulate the extracellular signal-regulated kinase (ERK), leading to various forms of plasticity in the brain. Little is known, however, on the in vivo short- and long-term activation and regulation of the components of mitogen-activated protein kinase (MAPK)/ERK signaling by CB. The CB agonist WIN55212-2 (8 mg/kg) increased the immunodensities of phosphorylated c-Raf-1 (42%), MEK1/2 (63%), ERK1 (24%), and ERK2 (28%) in the rat cerebral frontal cortex. These effects were antagonized by SR141716A (rimonabant, 10 mg/kg), a selective CB(1) receptor antagonist. Repeated WIN55212-2 treatment (2-8 mg/kg for 5 days) resulted in tachyphylaxis to the acute activation of Raf-MEK-ERK signaling. Acute WIN55212-2 also induced a hypothermic effect in rats, which was reduced after repeated administration (tolerance). Treatment with SR141716A after chronic WIN55212-2 resulted in the expected cannabinoid withdrawal syndrome, without concomitant alterations in the phosphorylation state of c-Raf-1, MEK1/2, or ERK1/2. Pretreatment with SL327 (20 mg/kg, a MEK1/2 inhibitor) increased the basal phosphorylation of c-Raf-1 (40%) and MEK1/2 (74%; feedback regulation) and fully prevented the up-regulation of ERK1/2 (23-31%) induced by WIN55212-2. Pretreatment with MK801 (1 mg/kg, a NMDA receptor antagonist) effectively blocked the up-regulation c-Raf-1 (41%), MEK1/2 (57%) and ERK1/2 (25-30%) induced by the CB agonist. The main findings demonstrate that the acute stimulation of CB(1) receptors in the frontal cortex results in the sequential phosphorylation of Raf-MEK-ERK cascade, in which c-Raf-1 activation (rate-limiting process) plays a crucial role. Moreover, the in vivo stimulating effect of WIN55212-2 on Raf-MEK-ERK signaling is under the extrinsic regulation of an excitatory glutamatergic mechanism.     

ERK1/2 and ERK5 have distinct roles in the regulation of brain-derived neurotrophic factor expression

Neurotrophins play essential roles in the development, differentiation, and survival of neuronal and nonneuronal cells. Alterations in neurotrophin expression have been implicated in a variety of neurodegenerative disorders. Dysregulation of brain-derived neurotrophic factor (BDNF) has been implicated in deficits of long-term potentiation and cognition and may contribute to the development of Alzheimer's disease (AD). In this study, we used complementary pharmacological and molecular approaches to evaluate the role of ERK1/2 and ERK5, two members of the MAPK pathway associated with neuroprotection, in regulating BDNF expression in C6 glial cells and primary astrocytes. Our data revealed that U0126, an inhibitor of both ERK5 and ERK1/2, increased the levels of BDNF mRNA, whereas the MEK1/2-specific inhibitor PD184352 did not, suggesting that ERK5 exerts negative control over BDNF expression. This was supported by experiments in which RNAi-mediated depletion of ERK5 led to an increase in BDNF. In contrast, transfection with constitutively active MEK5 resulted in an inhibition of BDNF expression, confirming the inhibitory role of ERK5 in the regulation of BDNF. Interestingly, transfection with the dominant active mutant of MEK1 (MEKR4F), the upstream activator of ERK1/2, resulted in a modest increase in BDNF levels. Collectively, our data suggest that ERK5 and ERK1/2 exert opposite effects on BDNF expression and support the hypothesis that an imbalance of these two signaling pathways may contribute to the pathology of diseases in which neurotrophin dysregulation is noted.