Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.     

Activation of mGlu3 Receptors Stimulates the Production of GDNF in Striatal Neurons

Metabotropic glutamate (mGlu) receptors have been considered potential targets for the therapy of experimental parkinsonism. One hypothetical advantage associated with the use of mGlu receptor ligands is the lack of the adverse effects typically induced by ionotropic glutamate receptor antagonists, such as sedation, ataxia, and severe learning impairment. Low doses of the mGlu2/3 metabotropic glutamate receptor agonist, {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 (0.25–3 mg/kg, i.p.) increased glial cell line-derived neurotrophic factor (GDNF) mRNA and protein levels in the mouse brain, as assessed by in situ hybridization, real-time PCR, immunoblotting, and immunohistochemistry. This increase was prominent in the striatum, but was also observed in the cerebral cortex. GDNF mRNA levels peaked at 3 h and declined afterwards, whereas GDNF protein levels progressively increased from 24 to 72 h following {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 injection. The action of {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 was abrogated by the mGlu2/3 receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495 (1 mg/kg, i.p.), and was lost in mGlu3 receptor knockout mice, but not in mGlu2 receptor knockout mice. In pure cultures of striatal neurons, the increase in GDNF induced by {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 required the activation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways, as shown by the use of specific inhibitors of the two pathways. Both in vivo and in vitro studies led to the conclusion that neurons were the only source of GDNF in response to mGlu3 receptor activation. Remarkably, acute or repeated injections of {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 at doses that enhanced striatal GDNF levels (0.25 or 3 mg/kg, i.p.) were highly protective against nigro-striatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice, as assessed by stereological counting of tyrosine hydroxylase-positive neurons in the pars compacta of the substantia nigra. We speculate that selective mGlu3 receptor agonists or enhancers are potential candidates as neuroprotective agents in Parkinson''s disease, and their use might circumvent the limitations associated with the administration of exogenous GDNF.     

The potent, selective mGlu2/3 receptor agonist LY379268 increases extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid in the medial prefrontal cortex of the freely moving rat

Previous work has shown that the potent, selective metabotropic glutamate mGlu2/3 receptor agonist LY379268 acts like the atypical antipsychotic clozapine in behavioral assays. To investigate further the potential antipsychotic actions of this agent, we examined the effects of LY379268 using microdialysis in awake, freely moving rats, on extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in rat medial prefrontal cortex. Systemic LY379268 increased extracellular levels of dopamine, DOPAC, HVA, and 5-HIAA in a dose-dependent, somewhat delayed manner. LY379268 (3 mg/kg s.c. ) increased levels of dopamine, DOPAC, HVA, and 5-HIAA to 168, 170, 169, and 151% of basal, respectively. Clozapine (10 mg/kg) also increased dopamine, DOPAC, and HVA levels, with increases of 255, 262, and 173%, respectively, but was without effect on extracellular 5-HIAA levels by 3 mg/kg LY379268 were reversed by the selective mGlu2/3 receptor antagonist LY341495 (1 mg/kg). Furthermore, LY379268 (3 mg/kg)-evoked increases in DOPAC and HVA were partially blocked and the increase in 5-HIAA was completely blocked by local application of 3 microM tetrodotoxin. Therefore, we have demonstrated that mGlu2/3 receptor agonists activate dopaminergic and serotonergic brain pathways previously associated with the action of atypical antipsychotics such as clozapine and other psychiatric agents.     

Intracellular signalings underlying bradykinin-induced matrix metalloproteinase-9 expression in rat brain astrocyte-1

Bradykinin (BK), an inflammatory mediator, has been shown to increase the expression of proteins such as matrix metalloproteinases (MMPs) on brain cells and contributes to the pathophysiology of inflammatory responses. However, the mechanisms regulating MMP-9 expression by BK in rat brain astrocytes-1 (RBA-1) remain unclear. Here we report that the mitogen-activated protein kinase (MAPK) and NF-kappaB pathways participate in the induction of MMP-9 expression induced by BK in RBA cells. Zymographic, Western blotting, and RT-PCR analyses showed that BK increased expression of MMP-9 mRNA and protein in a time- and concentration-dependent manner. BK-induced MMP-9 mRNA and protein expression was inhibited by MEK1/2 inhibitor PD98059, PI3-K inhibitor LY294002, and NF-kappaB inhibitor helenalin. In accordance with these findings, BK-induced phosphorylation of p42/p44 MAPK and Akt and activation of NF-kappaB was attenuated by prior treatment with PD98059, LY294002, and helenalin, respectively. The effects of BK on MMP-9 expression and p42/p44 MAPK and Akt phosphorylation were inhibited by B(2) receptor antagonist Hoe 140, indicating the involvement of B(2) receptors revealed by [(3)H]-BK binding assay. Furthermore, BK-stimulated translocation of NF-kappaB into the nucleus was revealed by Western blotting and immnofluorescence staining and blocked by Hoe140, PD98059, LY294002, and helenalin. Taken together, these results suggest that in RBA cells, activation of p42/p44 MAPK and Akt cascades mediated through NF-kappaB pathway are essential for BK-induced MMP-9 gene expression. This study may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation and brain astrocytoma.     

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.     

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis

Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose-dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl-inositol-3-kinase (PI-3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin-stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI-3K- and MEK-dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI-3K rather than MEK supports insulin-mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473-phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI-3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome-c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin-dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.     

Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells

17Beta-estradiol (E2) induces proliferation and c-fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at -325 and -296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4-SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src-ras-PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src-ras activation of both PI3-K- and MAPK-dependent signaling pathways.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268

Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs).     

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG‐63 cells

Insulin has been proposed to be an anabolic agent in bone, but the mechanisms underlying insulin effects on osteoblast differentiation are still not clear. To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line‐MG‐63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3‐K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG‐63 cells in a time‐ and dose‐dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3‐K inhibitor LY294002 significantly reversed the down‐regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up‐regulation of Osx and IGF‐1 expression after insulin treatment. Therefore, the data obtained from this study suggested that insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG‐63 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Pharmacologic inhibition of MEK and PI-3K converges on the mTOR/S6 pathway to decelerate cellular senescence

Inhibition of mTOR by rapamycin prevents cellular senescence. Here we investigated the effects of MEK and PI-3K on cellular senescence. Unlike LY294002 (PI-3K inhibitor), both U0126 and PD98059 (MEK inhibitors) did not significantly decrease beta-Gal staining in aging human fibroblasts and fibrosarcoma cells. However, using a sensitive, functional method, we identified that not only LY294002 but also U0126 prevented irreversible loss of proliferative potential associated with cellular senescence. At concentrations that blocked S6 phosphorylation, rapamycin, U0126 and LY294002 equally prevented senescence. Furthermore, there was no additive effect by combining of rapamycin with either U0126 or LY294002. Taken together this suggests that (a) simultaneous activation of PI-3K and MEK is required to ensure cellular senescence and (b) U0126 and LY294002 suppresses senescence via the rapamycin-sensitive pathway.     

Neuroprotection of Interleukin-6 Against NMDA-induced Neurotoxicity is Mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT Signaling Pathways

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca²⁺ fluorescence intensity, cytosolic Ca²⁺-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca²⁺ overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca²⁺ overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.     

Activation of multiple signaling pathways is critical for fibroblast growth factor 2- and vascular endothelial growth factor-stimulated ovine fetoplacental endothelial cell proliferation

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by N(G)-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.