62 research outputs found
Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of delta-opioid receptor
No full textWe have previously reported that N-desmethylclozapine (NDMC), a major clozapine metabolite, acts as a δ-opioid receptor agonist. Here, we show that in different cellular systems NDMC regulates protein kinase B/Akt (Akt) signaling through the activation of δ-opioid receptors. In Chinese hamster ovary cells transfected with the human δ-opioid receptor (CHO/DOR), NDMC induced a time- and concentration-dependent phosphorylation of Akt at Thr308 and glycogen synthase kinase-3β (GSK-3β) at Ser9 and these effects were fully blocked by the δ-opioid receptor antagonist naltrindole. NDMC-induced Akt and GSK-3β phosphorylations were completely prevented by pertussis toxin, the Src tyrosine kinase inhibitor PP2 and the selective insulin-like growth factor-I (IGF-I) receptor tyrosine kinase inhibitor tyrphostin AG 1024. NDMC stimulated IGF-I receptor β subunit tyrosine phosphorylation and this effect was prevented by either naltrindole or PP2. Blockade of phosphatidylinositol 3-kinase (PI3K) α, but not PI3Kγ, suppressed NDMC-induced Akt and GSK-3β phosphorylation, whereas inhibition of Akt curtailed the stimulation of GSK-3β phosphorylation. In rat nucleus accumbens, NDMC induced Akt and GSK-3β phosphorylation either in vitro or in vivo and these effects were prevented by naltrindole. NDMC also regulated Akt and GSK-3β phosphorylation through δ-opioid receptors in NG108-15 cells. In these cells NDMC counteracted oxidative stress-induced apoptosis and the effect was lost following PI3K inhibition. These data demonstrate that in different cell systems NDMC can stimulate Akt signaling by activating Gi/Go-coupled δ-opioid receptors, which, at least in CHO/DOR cells, regulate PI3Kα through Src-dependent transactivation of the IGF-I receptor, and indicate that through this mechanism NDMC can exert neuroprotective effects
Delta-opioid receptors stimulate GLUT1-mediated glucose uptake through Src- and IGF-1 receptor-dependent activation of PI3-kinase signalling in CHO cells
No full textAlthough opioids have been reported to affect glucose homeostasis, relatively little is known on the role of δ-opioid receptors. We have investigated the regulation of glucose transport by human δ-opioid receptors expressed in Chinese hamster ovary cells.
EXPERIMENTAL APPROACH:
The uptake of [(3)H]-2-deoxy-D-glucose and 3-O-[methyl-[(3)H]]-D-glucose in response to δ-opioid receptor ligands and the expression of GLUT1, GLUT3 and GLUT4 glucose transporters were examined. Moreover, the effects of intracellular signal transduction inhibitors on δ-opioid receptor-regulated [(3)H]-2-deoxy-D-glucose uptake and protein phosphorylation were investigated.
KEY RESULTS:
Activation of δ-opioid receptors rapidly stimulated [(3)H]-2-deoxy-D-glucose and 3-O-[methyl-[(3)H]]-D-glucose uptakes, which were blocked by the GLUT inhibitors cytochalasin B and phloretin. The stimulation of [(3)H]-2-deoxy-D-glucose uptake that occurred without a change in plasma membrane GLUT1 - required the coupling to G(i) /G(o) proteins - was independent of cAMP and extracellular signal-regulated protein kinases, and was suppressed by blockade of Src and insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinases. Inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin or LY294002 and by PI3Kα, but not γ, isoform-selective inhibitors greatly reduced the δ-opioid receptor stimulation of glucose uptake. Moreover, the response was attenuated by overexpressing a dominant-negative kinase-deficient Akt form and by chemical inhibition of Akt. Stimulation of δ-opioid receptors increased protein kinase Cζ/λ (PKCζ/λ) phosphorylation and a selective PKCζ/λ inhibitor slightly reduced opioid stimulation of glucose uptake.
CONCLUSIONS AND IMPLICATIONS:
δ-Opioid receptors stimulated glucose transport probably by enhancing GLUT1 intrinsic activity through a signalling cascade involving G(i)/G(o), Src, IGF-1R, PI3Kα, Akt and, to a minor extent, PKCζ/λ. This effect may contribute to the opioid regulation of glucose homeostasis in physio-pathological conditions
Oleoylethanolamide and Palmitoylethanolamide Enhance IFNβ-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells
No full textOleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are endogenous lipids that act as agonists of the peroxisome proliferator-activated receptor α (PPARα). Recently, an interest in the role of these lipids in malignant tumors has emerged. Nevertheless, the effects of OEA and PEA on human neuroblastoma cells are still not documented. Type I interferons (IFNs) are immunomodulatory cytokines endowed with antiviral and anti-proliferative actions and are used in the treatment of various pathologies such as different cancer forms (i.e., non-Hodgkin’s lymphoma, melanoma, leukemia), hepatitis B, hepatitis C, multiple sclerosis, and many others. In this study, we investigated the effect of OEA and PEA on human neuroblastoma SH-SY5Y cells treated with IFNβ. We focused on evaluating cell viability, cell proliferation, and cell signaling. Co-exposure to either OEA or PEA along with IFNβ leads to increased apoptotic cell death marked by the cleavage of caspase 3 and poly-(ADP ribose) polymerase (PARP) alongside a decrease in survivin and IKBα levels. Moreover, we found that OEA and PEA did not affect IFNβ signaling through the JAK-STAT pathway and the STAT1-inducible protein kinase R (PKR). OEA and PEA also increased the phosphorylation of p38 MAP kinase and programmed death-ligand 1 (PD-L1) expression both in full cell lysate and surface membranes. Furthermore, GW6471, a PPARα inhibitor, and the genetic silencing of the receptor were shown to lower PD-L1 and cleaved PARP levels. These results reveal the presence of a novel mechanism, independent of the IFNβ-prompted pathway, by which OEA and PEA can directly impair cell survival, proliferation, and clonogenicity through modulating and potentiating the intrinsic apoptotic pathway in human SH-SY5Y cells
Interferon-β alters neurotrophin-3 TrkC receptor expression and signaling in differentiated human SH-SY5Y neuroblastoma cells
No full textBoth type I interferons (IFNs) and neurotrophins act on neuronal cells to regulate survival, growth and differentiation. However, relatively little is known on the interaction between these two classes of regulatory proteins. We have previously reported that IFN-β causes down-regulation of the brain-derived neurotrophic factor/TrkB receptor function and up-regulation of the nerve growth factor/TrkA/p75 receptor complex. In the present study, we investigated the effects of long-term exposure to IFN-β on the functional activity of the neurotrophin 3 (NT3)/TrkC receptor system in differentiated SH-SY5Y human neuroblastoma cells. We found that IFN-β treatment curtailed NT3-induced activation of distinct signaling molecules regulated by TrkC receptor, including protein kinase B/Akt, phospholipase Cγ1 and extracellular signal-regulated kinase 1 and 2. Analysis of TrkC receptor expression showed the presence of both 140 kDa full-length and 90-100 kDa kinase-deficient truncated isoforms. Exposure to IFN-β induced a predominant enhancement in the levels of the truncated TrkC receptor isoform in a time-dependent fashion. As the main function of the truncated receptor is the inhibition of the kinase-active TrkC receptor isoforms, our data suggest that long-term exposure to IFN-β down-regulates NT3 signaling by altering the balance between kinase-active and inactive TrkC receptor isoforms
Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis
No full textHistone deacetylase (HDAC) inhibitors are novel chemotherapy agents with potential utility in the treatment of neuroblastoma, the most frequent solid tumor of childhood. Previous studies have shown that the exposure of human neuroblastoma cells to some HDAC inhibitors enhanced the expression of the common neurotrophin receptor p75NTR. In the present study we investigated whether the upregulation of p75NTR could be exploited to render neuroblastoma cells susceptible to the cytotoxic action of an anti-p75NTR antibody conjugated to the toxin saporin-S6 (p75IgG-Sap). We found that two well-characterized HDAC inhibitors, valproic acid (VPA) and entinostat, were able to induce a strong expression of p75NTR in different human neuroblastoma cell lines but not in other cells, with entinostat, displaying a greater efficacy than VPA. Cell pretreatment with entinostat enhanced p75NTR internalization and intracellular saporin-S6 delivery following p75IgG-Sap exposure. The addition of p75IgG-Sap had no effect on vehicle-pretreated cells but potentiated the apoptotic cell death that was induced by entinostat. In three-dimensional neuroblastoma cell cultures, the subsequent treatment with p75IgG-Sap enhanced the inhibition of spheroid growth and the impairment of cell viability that was produced by entinostat. In athymic mice bearing neuroblastoma xenografts, chronic treatment with entinostat increased the expression of p75NTR in tumors but not in liver, kidney, heart, and cerebellum. The administration of p75IgG-Sap induced apoptosis only in tumors of mice that were pretreated with entinostat. These findings define a novel experimental strategy to selectively eliminate neuroblastoma cells based on the sequential treatment with entinostat and a toxin-conjugated anti-p75NTR antibody
M3 MUSCARINIC ACETYLCHOLINE RECEPTORSACTIVATE THE METABOLIC SENSOR AMP-ACTIVATEDPROTEIN KINASE AND STIMULATE GLUCOSE UPTAKETHROUGH STORE-OPERATED CA2+ ENTRY IN SH-SY5YHUMAN NEUROBLASTOMA CELLS
No full textGq/11-coupled muscarinic acetylcholine receptors (mAChRs) expressedin peripheral tissues have been recently reported to regulate energy metabolism and maintenance of physiological blood glucose levels.However, relatively little is known on the ability of these receptors tocontrol energy homeostasis in neuronal cells. Previous studies haveshown that in human SH-SY5Y neuroblastoma cells mAChRs stimu-late the metabolic sensor AMP-activated protein kinase (AMPK)through Ca2+/calmodulin-dependent protein kinase kinase-b (CaM-KKb), but the source of Ca2+required for this response has not yetbeen elucidated. The present study investigates the involvement ofstore-operated Ca2+entry (SOCE) in mAChRs-induced AMPK activa-tion. We found that in SH-SY5Y cells AMPK activation was mediatedby pharmacologically defined M3mAChRs, strictly dependent onextracellular Ca2+and blocked by the SOCE inhibitor 2-amin-oethoxydiphenyl borate (2-APB). Stimulation of M3mAChR activatedthe endoplasmic reticulum Ca2+sensor stromal interaction molecule(STIM) 1, as indicated by the redistribution of STIM1 immunofluores-cence into puncta, and promoted the association of STIM1 with twocomponents of SOCE channels, transient receptor potential canonical 1(TRPC1) and Orai1. Either STIM1 or TRPC1 knockdown curtailed M3mAChR-induced AMPK activation. Moreover, Ca2+store depletion bythapsigargin or cyclopiazonic acid activated AMPK in a manner simi-lar to M3-mAChRs. We also found that activation of M3mAChRsstimulated glucose uptake and this response required extracellularCa2+, was inhibited by 2-APB and reduced by knockdown of STIM1.Moreover, pharmacological inhibition of either CaMKKß withSTO609 or AMPK with Compound C prevented M3mAChR-stimu-lated glucose uptake. The data provide evidence that in a neuronal-likecell system activation of M3mAChRs stimulate AMPK and down-stream glucose uptake by triggering SOCE and suggest that SOCE is acritical process linking M3AChRs to the control of neuronal energy metabolis
The Orphan G Protein-coupled Receptor 75 Signaling is Activated by the Chemokine CCL5
No full textThe chemokine CCL5 prevents neuronal cell death mediated both by amyloid β, as well as the human immunodeficiency virus (HIV) viral proteins gp120 and Tat. Because CCL5 binds to CCR5, CCR3 and/or CCR1 receptors, it is unclear which of these receptors plays a role in neuroprotection. Indeed, CCL5 also has neuroprotective activity in cells lacking these receptors. CCL5 may bind to a G protein-coupled receptor 75 (GPR75), which encodes for a 540 amino-acid orphan receptor of the Gqα family. In this study, we have used SH-SY5Y human neuroblastoma cells to characterize whether CCL5 could activate a Gq signaling through GPR75. Both qPCR and flow cytometry show that these cells express GPR75 but do not express CCR5, CCR3 or CCR1 receptors. SY-SY5Y cells were then used to examine CCL5-mediated signaling. We report that CCL5 promotes a time- and concentration-dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3β and extracellular signal-regulated kinase (ERK) 1/2. Specific antagonists of CCR5, CCR3 and CCR1 did not prevent CCL5 from increasing phosphorylated AKT or ERK. Moreover, CCL5 promotes a time-dependent internalization of GPR75. Lastly, knocking down GPR75 expression by a CRISPR-Cas9 approach inhibited the ability of CCL5 to activate pERK in SH-SY5Y cells. Therefore, we propose that GPR75 is a novel receptor for CCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small molecule GPR75 agonists that mimic CCL5
Production of interferon-β (INF-β) in E. coli and strategies for improving its bioavailability and pharmacological profile.
No full textIFN-β is the main therapeutic agent which convincingly reduces multiple sclerosis relapse rate and retards disability. IFN-β, expressed both in mammalian and bacterial cells, is used in therapy. However, even if costs and yield in E. coli are very advantageous, its production is extremely difficult, due to its toxicity and extreme insolubility. Bio-Ker set up a thermoinducible expression system with optimized codon usage, which allows the expression of a fusion protein reaching a yield up to 2mg/l. Biological activity of the purified protein was evaluated by measuring inhibition of cell multiplication, and phosphorylation of Stat1 in Hela cells. Principal issues related to IFN-β are protein aggregation, with loss of bioavailability up to 70%, and high immunogenicity, with production of neutralizing Ab which interrupt therapy. In order to ameliorate the bio-similar product, Bio-Ker produced some site-specific muteins, according to molecular modelling design. Muteins have been evaluated for in vitro and in vivo pharmacological profiles. Moreover, IFN-β naturally exhibits a relatively short serum half-life, and we are planning to obtain a long lasting IFN-β mutein by conjugation to polymers which will entrap the protein in pharmaceutical controlled release devices to increase their bioavailability and to improve their pharmacological profile. Pharmacological, pharmacokinetic and pharmacodynamic data will be discussed
CCL5 activates a orphan G-protein coupled receptor 75 Neuroprotective effect of CCL5 via the G-protein coupled receptor 75 (GPR75) activation. 20th Meeting of Society of NeuroImmune Pharmacology (SNIP). 26-29 March, 2014 New Orleans, U.S.A.
No full textThe chemokine CCL5 affects the binding of the envelop protein gp120 to the co-receptor CCR5. CCL5 also
prevents neuronal cell death mediated by the X4 gp120 and Tat, which have no affinity for CCR5. The mechanism
of action of this chemokine remains to be fully characterized. Recent studies and preliminary data have shown that
CCL5 activates a GPR75 which belongs to the Gqα family. This receptor is more abundant in the brain than in the
immune organs. Moreover, CCL5 activates various pro-survival signaling molecules, including inositol
triphosphate, phosphatidylinositol 3-kinase and its downstream targets, protein kinase B (Akt) and extracellular
signal-regulated kinases (ERK1/2), in SH-SY5Y human neuroblastoma cells. These cells do not express CCR5,
CCR3 and CCR1, receptors known to bind to CCL5. Moreover, CCL4, CCL7 and CCL3, other chemokines that
bind to CCR5, CCR3 and CCR1, failed to activate these signaling molecules in SH-SY5Y cells. Akt and ERK1/2
phosphorylation were blocked by the Wortmannin and U73122, inhibitors of Akt and ERK1/2, respectively. At the
same time these responses were insensitive to pertussis toxin, a Gi inhibitor, suggesting that CCL5 activates a
GPCR coupled to Gq proteins. Therefore, GPR75 could explain the neuroprotective activity of CCL5 against gp120
and Tat. The discovery and characterization of compounds that prevent or limit the neurodegeneration that follows
HIV infection of the brain is a great challenge for HIV research. Thus, the results provide new mechanistic insight
which can be instrumental in addressing this challenge. Supported by NS 079172, NS 074916, DA 03228
Downregulation of TrkB expression and signaling by valproic acid and other histone deacetylase inhibitors
No full textValproic acid (VPA) has been shown to regulate the levels of brain-derived neurotrophic factor (BDNF), but it is not known whether this drug can affect the neuronal responses to BDNF. In the present study, we show that in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, prolonged exposure to VPA reduces the expression of the BDNF receptor TrkB at the protein and mRNA levels and inhibits the intracellular signaling, neurotrophic activity, and prosurvival function of BDNF. VPA downregulates TrkB and curtails BDNF-induced signaling also in differentiated Kelly and LAN-1 neuroblastoma cells and primary mouse cortical neurons. The VPA effect is mimicked by several histone deacetylase (HDAC) inhibitors, including the class I HDAC inhibitors entinostat and romidepsin. Conversely, the class II HDAC inhibitor MC1568, the HDAC6 inhibitor tubacin, the HDAC8 inhibitor PCI-34051, and the VPA derivative valpromide have no effect. In neuroblastoma cells and primary neurons both VPA and entinostat increase the cellular levels of the transcription factor RUNX3, which negatively regulates TrkB gene expression. Treatment with RUNX3 siRNA attenuates VPA-induced RUNX3 elevation and TrkB downregulation. VPA, entinostat, HDAC1 depletion by siRNA, and 3-deazaneplanocin A (DZNep), an inhibitor of the polycomb repressor complex 2 (PRC2), decrease the PRC2 core component EZH2, a RUNX3 suppressor. Like VPA, HDAC1 depletion and DZNep increase RUNX3 and decrease TrkB expression. These results indicate that VPA downregulates TrkB through epigenetic mechanisms involving the EZH2/RUNX3 axis and provide evidence that this effect implicates relevant consequences with regard to BDNF efficacy in stimulating intracellular signaling and functional responses.SIGNIFICANCE STATEMENTThe tropomyosin-related kinase receptor B (TrkB) mediates the stimulatory effects of brain-derived neurotrophic factor (BDNF) on neuronal growth, differentiation, and survival and is highly expressed in aggressive neuroblastoma and other tumors. Here we show that exposure to valproic acid (VPA) downregulates TrkB expression and functional activity in retinoic acid-differentiated human neuroblastoma cell lines and primary mouse cortical neurons. The effects of VPA are mimicked by other histone deacetylase (HDAC) inhibitors and HDAC1 knockdown and appear to be mediated by an epigenetic mechanism involving the upregulation of RUNX3, a suppressor of TrkB gene expression. TrkB downregulation may have relevance for the use of VPA as a potential therapeutic agent in neuroblastoma and other pathologies characterized by an excessive BDNF/TrkB signaling
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