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.     

Neurite outgrowth of NG108-15 cells induced by heat shock protein 90 inhibitors

We previously reported that radicicol (Rad) and geldanamycin (Geld), heat shock protein 90 (Hsp90) inhibitors, potentiate neurite growth of cultured sensory neurons from chick embryo. We now show that the antibiotics induce neurite growth in NG108-15 cells. Treatment of the cells with these drugs caused transient decrease in protein levels of Raf1, ERK1/2, phosphorylated ERK1/2, Akt1, and CDK4. The neurite growth of NG108-15 induced by the inhibitors was blocked by actynomycin D, but the neurite growth stimulated by dbcAMP in the cells was not affected. The neurite growth could be due to a change in the synthesis of some specific protein(s) and is speculated to be due to the transient downregulation of particular-signaling molecules stabilized by Hsp90.     

Proximal events in signaling by plasma membrane estrogen receptors

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.     

Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.     

Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Kinase suppressor of Ras 1 (KSR1) and KSR2 are scaffolds that promote extracellular signal-regulated kinase (ERK) signaling but have dramatically different physiological functions. KSR2(-/-) mice show marked deficits in energy expenditure that cause obesity. In contrast, KSR1 disruption has inconsequential effects on development but dramatically suppresses tumor formation by activated Ras. We examined the role of KSR2 in the generation and maintenance of the transformed phenotype in KSR1(-/-) mouse embryo fibroblasts (MEFs) expressing activated Ras(V12) and in tumor cell lines MIN6 and NG108-15. KSR2 rescued ERK activation and accelerated proliferation in KSR1(-/-) MEFs. KSR2 expression alone induced anchorage-independent growth and synergized with the transforming effects of Ras(V12). Similarly, RNA interference (RNAi) of KSR2 in MIN6 and NG108-15 cells inhibited proliferation and colony formation, with concomitant defects in AMP-activated protein kinase (AMPK) signaling, nutrient metabolism, and metabolic capacity. While constitutive activation of AMPK was sufficient to complement the loss of KSR2 in metabolic signaling and anchorage-independent growth, KSR2 RNAi, MEK inhibition, and expression of a KSR2 mutant unable to interact with ERK demonstrated that mitogen-activated protein (MAP) kinase signaling is dispensable for the transformed phenotype of these cells. These data show that KSR2 is essential to tumor cell energy homeostasis and critical to the integration of mitogenic and metabolic signaling pathways.     

Calcium activation of ERK mediated by calmodulin kinase I

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.     

Delayed transactivation of the receptor for nerve growth factor is required for sustained signaling and differentiation by alpha2-adrenergic receptors in transfected PC12 cells

Alpha2-adrenergic receptors have been reported to induce subtype-specific neuronal differentiation in vitro, but the signaling mechanisms that mediate this effect have not been characterized. In the present study we found that stimulated alpha2-ARs induce delayed transactivation of TrkA in PC12 cells. The transactivation of TrkA was sensitive to the PP1 inhibitor of the Src family kinases and required prior transactivation of the EGF receptor. Moreover, alpha2-adrenergic receptors induced sustained activation of MAPK and Akt. The sustained activation of Akt, but not of MAPK, was subtype-specific and correlated with the neuronal differentiation of PC12 cells, with the order alpha2A相似文献   

Different effects of five dopamine receptor subtypes on nuclear factor-kappaB activity in NG108-15 cells and mouse brain

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-kappaB activity. To investigate whether other dopamine receptor subtypes regulate NF-kappaB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-kappaB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-kappaB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element-luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-kappaB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-kappaB activity. By contrast, risperidone treatment enhanced NF-kappaB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-kappaB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-kappaB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.     

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma × glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the “pro-survival” kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma × glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen^2,5]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G_i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G_q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.     

Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems.

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.     

Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction

Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones.     

Overexpression of Adhesion Molecule L1 in NG108-15 Neuroblastoma × Glioma Hybrid Cells Enhances Dibutyryl Cyclic AMP-Induced Neurite Outgrowth and Functional Synapse Formation with Myotubes

Abstract: The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma × glioma hybrid NG108-15 cells. L1 overexpression was found in ∼50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 m M dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4–7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.     

Nitric oxide inhibits the expression of AT1 receptors in neurons

We have previously observed an increased of angiotensin II (ANG II) type 1 receptor (AT(1)R) with enhanced AT(1)R-mediated sympathetic outflow and concomitant downregulation of neuronal nitric oxide (NO) synthase (nNOS) with reduced NO-mediated inhibition from the paraventricular nucleus (PVN) in rats with heart failure. To test the hypothesis that NO exerts an inhibitory effect on AT(1)R expression in the PVN, we used primary cultured hypothalamic cells of neonatal rats and neuronal cell line NG108-15 as in vitro models. In hypothalamic primary culture, NO donor sodium nitroprusside (SNP) induced dose-dependent decreases in mRNA and protein of AT(1)R (10(-5) M SNP, AT(1)R protein was 10 ± 2% of control level) while NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) induced dose-dependent increases in mRNA and protein levels of AT(1)R (10(-5) M l-NMMA, AT(1)R protein was 148 ± 8% of control level). Similar effects of SNP and l-NMMA on AT(1)R expression were also observed in NG108-15 cell line (10(-6) M SNP, AT(1)R protein was 30 ± 4% of control level while at the dose of 10(-6) M l-NMMA, AT(1)R protein was 171 ± 15% of the control level). Specific inhibition of nNOS, using antisense, caused an increase in AT(1)R expression while overexpression of nNOS, using adenoviral gene transfer (Ad.nNOS), caused an inhibition of AT(1)R expression in NG108 cells. Antisense nNOS transfection augmented the increase while Ad.nNOS infection blunted the increase in intracellular calcium concentration in response to ANG II treatment in NG108 cells. In addition, downregulation of AT(1)R mRNA as well as protein level in neuronal cell line in response to S-nitroso-N-acetyl pencillamine (SNAP) treatment was blocked by protein kinase G (PKG) inhibitor, while the peroxynitrite scavenger deforxamine had no effect. These results suggest that NO acts as an inhibitory regulator of AT(1)R expression and the activation of PKG is the required step in the regulation of AT(1)R gene expression via cGMP-dependent signaling pathway.     

Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.     

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.