Endogenous and exogenous fibroblast growth factor 2 support survival of chick retinal neurons by control of neuronal neuronal bcl-x(L) and bcl-2 expression through a fibroblast berowth factor receptor 1- and ERK-dependent pathway

Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-x(L) and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-x(L) and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-x(L) and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-x(L) levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-x(L), through ERK activation in retinal neurons.     

ERK 1/2 signaling pathway is involved in nicotine-mediated neuroprotection in spinal cord neurons

Evidence indicates that agonists of neuronal nicotinic receptors (nAChRs), including nicotine, can induce neuroprotective and anti-apoptotic effects in the CNS. To study these mechanisms, the present study focused on nicotine-mediated modulation of the extracellular regulated kinase 1 and 2 (ERK1/2) pathway in cultured spinal cord neurons. Exposure to nicotine (0.1-10 microM) for as short as 1 min markedly upregulated levels of phosphorylated ERK1/2 (pERK1/2) and increased total ERK1/2 activity. Inhibition studies with mecamylamine and alpha-bungarotoxin revealed that these effects were mediated by the alpha7 nicotinic receptor. In addition, pre-exposure to U0126, a specific inhibitor of the ERK1/2 signaling, prevented nicotine-mediated anti-apoptotic effects. To indicate if treatment with nicotine also can activate ERK1/2 in vivo, a moderate spinal cord injury (SCI) was induced in rats using a weight-drop device and nicotine was injected 2 h post-trauma. Consistent with in vitro data, nicotine increased levels of pERK1/2 in this animal model of spinal cord trauma. Results of the present study indicate that the ERK1/2 pathway is involved in anti-apoptotic effects of nicotine in spinal cord neurons and may be involved in therapeutic effects of nicotine in spinal cord trauma.     

Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase

Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.     

PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.

Expression of oncogenic ras in PC12 cells causes neuronal differentiation and sustained protein tyrosine phosphorylation and activity of extracellular signal-regulated kinases (ERKs), p42erk2 and p44erk1. Oncogenic N-ras-induced neuronal differentiation is inhibited by compounds that block ERK protein tyrosine phosphorylation or ERK activity, indicating that ERKs are not only activated by p21ras but serve as the primary downstream effectors of p21ras. Treatment of PC12 cells with nerve growth factor or fibroblast growth factor results in neuronal differentiation and in a sustained elevation of p21ras activity, of ERK activity, and of ERK tyrosine phosphorylation. Epidermal growth factor, which does not cause neuronal differentiation, stimulates only transient (< 1 hr) activation of p21ras and ERKs. These data indicate that transient activation of p21ras and, consequently, ERKs is not sufficient for induction of neuronal differentiation. Prolonged ERK activity is required: a consequence of sustained activation of p21ras by the growth factor receptor protein tyrosine kinase.     

Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala², D-Leu⁵]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH₃ revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.     

Distinct roles for ERK1 and ERK2 in pathophysiology of CNS

Mitogen-activated protein kinases ERK1 and ERK2 have been implicated in various pathophysiological events of the CNS,but their specific roles in cell processes under physiologic and pathological condit...     

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18‐1

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal‐regulated kinase (ERK) that mediates CB1R‐ and mGluR2/3‐induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock‐induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18‐1. Mimicking constitutive phosphorylation of Munc18‐1 results in a drastic decrease in synaptic transmission. ERK‐mediated phosphorylation of Munc18‐1 ultimately leads to degradation by the ubiquitin–proteasome system. Conversely, preventing ERK‐dependent Munc18‐1 phosphorylation increases synaptic strength. CB1R‐ and mGluR2/3‐induced synaptic inhibition and depolarization‐induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK‐dependent Munc18‐1 phosphorylation is blocked. Thus, ERK‐dependent Munc18‐1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.     

Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells represent an ideal source for cell engraftment in the damaged central nervous system (CNS). Understanding key signals that control ES cell differentiation may improve cell type-specific differentiation that is suitable for transplantation therapy. We tested the hypothesis that extracellular signal-regulated kinase (ERK) 1/2 phosphorylation is an early signaling event required for the neuronal differentiation of ES cells. Cultured mouse ES cells were treated with an all-trans-retinoic-acid (RA) protocol to generate neurally induced progenitor cells. Western blot analysis showed a dramatic increase in ERK 1/2 phosphorylation (p-ERK 1/2) 1-5 days after RA induction, which was attenuated in the presence of the p-ERK 1/2-specific inhibitor UO126. Phospho-ERK 1/2 inhibition significantly reduced the number of NeuN-positive cells and the expression of associated cytoskeletal proteins. In differentiating ES cells, there was increased nuclear translocation of STAT3 and decreased protein expression levels of GDNF, BDNF and NGF. STAT3 translocation was attenuated by UO126. Finally, caspase-3 activation was observed in the presence of UO126, suggesting that the ERK pathway also contributes to the survival of differentiating ES cells. These data indicate that ERK 1/2 phosphorylation is a key event required for early neuronal differentiation and survival of ES cells.     

Mitogen-activated protein kinase signaling is involved in suramin-induced neurite outgrowth in a neuronal cell line

Suramin is a well-known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers. Previous study showed that suramin is an activator of extracellular signal-regulated kinase (ERK1/2) signaling in several cell lines including Chinese hamster ovary cells, although the physiological relevance of this activation remains uncertain. Here, it was shown that suramin enhances neurite outgrowth concomitant with activation of ERK1/2 in Neuro-2a cells, a neuronal cell line. These neurite outgrowth and ERK1/2 activation were significantly inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase, as well as by activation of endogenous adenosine A2A receptors. The suramin-induced phosphorylation of ERK1/2 was also inhibited by inhibitors of Src family kinases. This attenuation of ERK1/2 activity was accompanied by a significant decrease in suramin-induced neurite outgrowth. These results suggest that suramin activates the Src/ERK1/2 signaling pathway that induces neurite outgrowth, both of which are negatively regulated by cAMP produced in response to activation of endogenous adenosine A2A receptors.     

Extracellular zinc triggers ERK-dependent activation of Na+/H+ exchange in colonocytes mediated by the zinc-sensing receptor

Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+ exchange in colonocytes. We demonstrate that Ca2+ release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+ but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by approximately 90% inhibition of the Zn2+ -dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+ and ERK1/2 activation. Application of both the IP3 pathway and PI 3-kinase antagonists largely inhibited Zn2+ -dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+ -dependent activation of ERK1/2 was addressed by monitoring Na+/H+ exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+ exchange, which was eliminated by cariporide (0.5 microm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+ or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+ -dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+ -dependent activation of H+ extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation.     

ERK1/2-p90RSK-mediated phosphorylation of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.     

4-O-Methylhonokiol attenuates memory impairment in presenilin 2 mutant mice through reduction of oxidative damage and inactivation of astrocytes and the ERK pathway

Presenilin 2 (PS2) mutation increases Aβ generation and neuronal cell death in the brains of Alzheimer disease (AD) patients. In a previous study, we showed that increased oxidative damage and activation of extracellular signal-regulated kinase (ERK) were associated with Aβ generation and neuronal cell death in neuronal cells expressing mutant PS2. In this study, we show that oral treatment with 4-O-methylhonokiol, a novel compound isolated from Magnolia officinalis, for 3 months (1.0mg/kg) prevented PS2 mutation-induced memory impairment and neuronal cell death accompanied by a reduction in Aβ(1-42) accumulation. We also found that 4-O-methylhonokiol inhibited PS2 mutation-induced activation of ERK and β-secretase, and oxidative protein and lipid damage, but recovered glutathione levels in the cortex and hippocampus of PS2 mutant mice. Additionally, 4-O-methylhonokiol prevented PS2 mutation-induced activation of astrocytes as well as production of TNF-α, IL-1β, reactive oxygen species (ROS), and nitric oxide (NO) in neurons. Generation of TNF-α, IL-1β, ROS, and NO and ERK activation in cultured astrocytes treated with lipopolysaccharide (1μg/ml) were also prevented by 4-O-methylhonokiol in a dose-dependent manner. These results suggest that the improving effects of 4-O-methylhonokiol on memory function may be associated with a suppression of the activation of ERK and astrocytes as well as a reduction in oxidative damage. Thus, 4-O-methylhonokiol may be useful in the prevention and treatment of AD.     

Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells

Insulin signalling is well studied in peripheral tissue, but not in neuronal tissue. To gain more insight into neuronal insulin signalling we examined protein kinase B (PKB) and extracellular regulated kinase 1 and 2 (ERK1/2) regulation in serum-deprived Neuro2a cells. Insulin phosphorylated PKB in a dose-dependent manner but reduced phosphorylation of ERK1/2. Both processes were phosphatidylinositol 3-kinase (PI3K) dependent. Interestingly, blockade of PI3K in combination with insulin induced phosphorylation of ERK1/2. The phosphorylation of ERK1/2 could be blocked with a specific inhibitor of mitogen-activated protein/ERK kinase (MEK), suggesting that it was mediated through the highly conserved Ras-Raf-MEK-ERK1/2 pathway. Prolonged exposure to high concentrations of insulin resulted in a desensitized PI3K-PKB route. The insulin-induced inhibition of ERK1/2 phosphorylation was also diminished when the PI3K-PKB route was desensitized. Blockade of PI3K in combination with insulin, however, still resulted in an unaltered MEK-dependent phosphorylation of ERK1/2. We conclude that PI3K is an important integrator of insulin signalling in Neuro2a cells as it regulates activation of PKB and inhibition of ERK1/2, and is sensitive to the duration of the insulin stimulus.