Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants.

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and -LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and -LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO-K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and -LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors.

Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis.

Histone 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.

The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid.

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.

Cannabinoid CB1 and CB2 receptors differentially regulate TNF-α-induced apoptosis and LPA1-mediated pro-survival signaling in HT22 hippocampal cells.

AIMS: The aim of the study was to investigate the interaction between cannabinoid CB1/CB2 and lysophosphatidic acid (LPA) receptors in controlling neuronal signaling and fate. METHODS: HT22 hippocampal cells were treated with different cannabinoid and LPA receptor agonists and antagonists. Western blot and immunofluorescence microscopy were used to study intracellular signaling and the expression of apoptotic markers. Cell viability was determined by a luminescence assay. KEY FINDINGS: Cannabinoid agonists induced activation of both ERK1/2 and p38 MAP kinases. The effects of the CB1/CB2 receptor agonist HU210 were antagonized by the CB1 antagonist rimonabant, whereas the responses to the CB2 agonist JWH133 were blocked by the CB2 antagonist SR144528. HU210 reduced the apoptotic cell death induced by the pro-inflammatory cytokine TNF-α, whereas JWH133 enhanced the cytokine cytotoxicity. Blockade of ERK1/2 and p38 MAPK activation abrogated the HU210 pro-survival and the JWH133 pro-apoptotic effects, respectively. HU210 and the endocannabinoid anandamide, but not JWH133, potentiated ERK1/2 stimulation by LPA and the tricyclic antidepressant amitriptyline acting through the LPA1 receptor. HU210 enhanced amitriptyline-stimulated CREB phosphorylation and protection against TNF-α-induced apoptosis, whereas JWH133 had no effect. ERK1/2 stimulation by either HU210 or amitriptyline was dependent on fibroblast growth factor receptor (FGF-R) kinase activity and the combination of the two stimulants induced FGF-R phosphorylation. Moreover, the CB1 receptor was found to co-immunoprecipitate with the LPA1 receptor. CONCLUSIONS: In HT22 hippocampal cells CB1 and CB2 receptors differentially regulate TNF-α-induced apoptosis and CB1 receptors positively interact with amitriptyline-stimulated LPA1 in promoting FGF-R-mediated ERK1/2 signaling and neuroprotection.

Valproic acid upregulates the expression of the p75NTR/sortilin receptor complex to induce neuronal apoptosis.

The antiepileptic and mood stabilizer agent valproic acid (VPA) has been shown to exert anti-tumour effects and to cause neuronal damage in the developing brain through mechanisms not completely understood. In the present study we show that prolonged exposure of SH-SY5Y and LAN-1 human neuroblastoma cells to clinically relevant concentrations of VPA caused a marked induction of the protein and transcript levels of the common neurotrophin receptor p75NTR and its co-receptor sortilin, two promoters of apoptotic cell death in response to proneurotrophins. VPA induction of p75NTR and sortilin was associated with an increase in plasma membrane expression of the receptor proteins and was mimicked by cell treatment with several histone deacetylase (HDAC) inhibitors. VPA and HDAC1 knockdown decreased the level of EZH2, a core component of the polycomb repressive complex 2, and upregulated the transcription factor CASZ1, a positive regulator of p75NTR. CASZ1 knockdown attenuated VPA-induced p75NTR overexpression. Cell treatment with VPA favoured proNGF-induced p75NTR/sortilin interaction and the exposure to proNGF enhanced JNK activation and apoptotic cell death elicited by VPA. Depletion of p75NTR or addition of the sortilin agonist neurotensin to block proNGF/sortilin interaction reduced the apoptotic response to VPA and proNGF. Exposure of mouse cerebellar granule cells to VPA upregulated p75NTR and sortilin and induced apoptosis which was enhanced by proNGF. These results indicate that VPA upregulates p75NTR apoptotic cell signalling through an epigenetic mechanism involving HDAC inhibition and suggest that this effect may contribute to the anti-neuroblastoma and neurotoxic effects of VPA.

Antidepressants induce profibrotic responses via the lysophosphatidic acid receptor LPA1.

Preclinical and clinical studies have indicated that antidepressants can promote inflammation and fibrogenesis, particularly in the lung, by mechanisms not fully elucidated. We have previously shown that different classes of antidepressants can activate the lysophosphatidic acid (LPA) receptor LPA1, a major pathogenetic mediator of tissue fibrosis. The aim of the present study was to investigate whether in cultured human dermal and lung fibroblasts antidepressants could trigger LPA1-mediated profibrotic responses. In both cell types amitriptyline, clomipramine and mianserin mimicked the ability of LPA to induce the phosphorylation/activation of extracellular signal -regulated kinases 1 and 2 (ERK1/2), which was blocked by the selective LPA1 receptor antagonist AM966 and the LPA1/3 antagonist Ki16425. Antidepressant-induced ERK1/2 stimulation was absent in fibroblasts stably depleted of LPA1 by short hairpin RNA transfection and was prevented by pertussis toxin, an uncoupler of receptors from Gi/o proteins. Like LPA, antidepressants stimulated fibroblasts proliferation and this effect was blocked by either AM966 or the MEK1/2 inhibitor PD98059. Moreover, by acting through LPA1 antidepressants induced the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, and caused an ERK1/2-dependent increase in the cellular levels of transforming growth factor-ß (TGF-ß)1, a potent fibrogenic cytokine. Pharmacological blockade of TGF-ß receptor type 1 prevented antidepressant- and LPA-induced α-SMA expression. These data indicate that in human dermal and lung fibroblasts different antidepressants can induce proliferative and differentiating responses by activating the LPA1 receptor coupled to ERK1/2 signalling and suggest that this property may contribute to the promotion of tissue fibrosis by these drugs.

Downregulation of TrkB Expression and Signaling by Valproic Acid and Other Histone Deacetylase Inhibitors.

Valproic 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 STATEMENT: The 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.

Inhibition of TNF-α-induced neuronal apoptosis by antidepressants acting through the lysophosphatidic acid receptor LPA1.

Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine considered to be implicated in the pathogenesis of major depressive disorder, is a critical regulator of neuronal cell fate. In the present study we found that TNF-α-induced apoptosis of HT22 hippocampal cells, a neuroblast-like cell line, was markedly attenuated by the antidepressants mianserin, mirtazapine and amitriptyline. The anti-apoptotic effect of the antidepressants was blocked by either pharmacological inhibition or gene silencing of the lysophosphatidic acid receptor LPA1. Mianserin failed to affect TNF-α-induced caspase 8 activation, but inhibited the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, procaspase 9 cleavage and downstream activation of caspase 3 in response to the cytokine. By acting through LPA1, mianserin also attenuated the enhanced pro-apoptotic response induced by the combination of TNF-α with other pro-inflammatory cytokines. TNF-α appeared to counterbalance its own pro-apoptotic response by activating NF-kB, ERK1/2 and JNK. Antidepressants had no significant effects on NF-kB activation, but potentiated the TAK-1-dependent phosphorylation of ERK1/2 and JNK elicited by the cytokine. This synergistic interaction was associated with enhanced JNK-mediated phosphorylation of Bcl-2 at Ser70 and increased ERK1/2-dependent mitochondrial accumulation of Mcl-1, two anti-apoptotic proteins that promote mitochondrial outer membrane stability. These results indicate that certain antidepressants, by activating LPA1 signalling, protect HT22 hippocampal cells from TNF-α-induced apoptosis through a mechanism involving, at least in part, the potentiation of the pro-survival pathways activated by the cytokine.

Muscarinic Acetylcholine Receptors Potentiate 5'-Adenosine Monophosphate-Activated Protein Kinase Stimulation and Glucose Uptake Triggered by Thapsigargin-Induced Store-Operated Ca2+ Entry in Human Neuroblastoma Cells.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca2+ entry (SOCE). However, whether mAChRs may control AMPK activity by regulating additional mechanisms beyond SOCE remains to be investigated. In the present study we examined the effects of mAChRs on AMPK when SOCE was induced by the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. We found that in SH-SY5Y cells depleted of Ca2+ by thapsigargin, the re-addition Ca2+ to the medium stimulated AMPK phosphorylation at Thr172, which is required for full kinase activity. This response occurred through SOCE, as it was blocked by either the SOCE modulator 2-aminoethoxydiphephenyl borate, knockdown of the SOCE molecular component STIM1, or inhibition of Ca2+/calmodulin (CaM)-dependent protein kinase kinase ß (CaMKKß). In thapsigargin-pretreated cells, stimulation of pharmacologically defined M3 mAChRs potentiated SOCE-induced AMPK activation. This potentiation did not involve an increased Ca2+ influx, but was associated with CaM mobilization from membrane to cytosol, increased CaM/CaMKKß interaction, and enhanced CaMKK stimulation by thapsigargin-induced SOCE. In thapsigargin-pretreated cells Ca2+ re-addition stimulated glucose uptake and increased the membrane expression of the glucose transporter GLUT1. Both responses were significantly potentiated by mAChRs. These data indicate that in human neuroblastoma cells mAChRs up-regulate AMPK and the downstream glucose uptake by triggering not only SOCE but also CaM translocation and enhanced formation of active CaM/CaMKKß complexes.

LPA1 is a key mediator of intracellular signalling and neuroprotection triggered by tetracyclic antidepressants in hippocampal neurons.

Both lysophosphatidic acid (LPA) and antidepressants have been shown to affect neuronal survival and differentiation, but whether LPA signalling participates in the action of antidepressants is still unknown. In this study, we examined the role of LPA receptors in the regulation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and neuronal survival by the tetracyclic antidepressants, mianserin and mirtazapine in hippocampal neurons. In HT22 immortalized hippocampal cells, antidepressants and LPA induced a time- and concentration-dependent stimulation of ERK1/2 phosphorylation. This response was inhibited by either LPA1 and LPA1/3 selective antagonists or siRNA-induced LPA1 down-regulation, and enhanced by LPA1 over-expression. Conversely, the selective LPA2 antagonist H2L5186303 had no effect. Antidepressants induced cyclic AMP response element binding protein phosphorylation and this response was prevented by LPA1 blockade. ERK1/2 stimulation involved pertussis toxin-sensitive G proteins, Src tyrosine kinases and fibroblast growth factor receptor (FGF-R) activity. Tyrosine phosphorylation of FGF-R was enhanced by antidepressants through LPA1 . Serum withdrawal induced apoptotic death, as indicated by increased annexin V staining, caspase activation and cleavage of poly-ADP-ribose polymerase. Antidepressants inhibited the apoptotic cascade and this protective effect was curtailed by blockade of either LPA1 , ERK1/2 or FGF-R activity. Moreover, in primary mouse hippocampal neurons, mianserin acting through LPA1 increased phospho-ERK1/2 and protected from apoptosis induced by removal of growth supplement. These data indicate that in neurons endogenously expressed LPA1 receptors mediate intracellular signalling and neuroprotection by tetracyclic antidepressants.

Interferon-ß Inhibits Neurotrophin 3 Signalling and Pro-Survival Activity by Upregulating the Expression of Truncated TrkC-T1 Receptor.

Although clinically useful for the treatment of various diseases, type I interferons (IFNs) have been implicated as causative factors of a number of neuroinflammatory disorders characterized by neuronal damage and altered CNS functions. As neurotrophin 3 (NT3) plays a critical role in neuroprotection, we examined the effects of IFN-ß on the signalling and functional activity of the NT3/TrkC system. We found that prolonged exposure of differentiated human SH-SY5Y neuroblastoma cells to IFN-ß impaired the ability of NT3 to induce transphosphorylation of the full-length TrkC receptor (TrkC-FL) and the phosphorylation of downstream signalling molecules, including PLCγ1, Akt, GSK-3ß and ERK1/2. NT3 was effective in protecting the cells against apoptosis triggered by serum withdrawal or thapsigargin but not IFN-ß. Prolonged exposure to the cytokine had little effects on TrkC-FL levels but markedly enhanced the messenger RNA (mRNA) and protein levels of the truncated isoform TrkC-T1, a dominant-negative receptor that inhibits TrkC-FL activity. Cell depletion of TrkC-T1 by small interfering RNA (siRNA) treatment enhanced NT3 signalling through TrkC-FL and allowed the neurotrophin to counteract IFN-ß-induced apoptosis. Furthermore, the upregulation of TrkC-T1 by IFN-ß was associated with the inhibition of NT3-induced recruitment of the scaffold protein tamalin to TrkC-T1 and tamalin tyrosine phosphorylation. These data indicate that IFN-ß exerts a negative control on NT3 pro-survival signalling through a novel mechanism involving the upregulation of TrkC-T1.

The GABAB positive allosteric modulators CGP7930 and GS39783 stimulate ERK1/2 signalling in cells lacking functional GABAB receptors.

The present study shows that the GABAB positive allosteric modulators (PAMs) CGP7930 and GS39783 stimulate extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signalling in cells that do not express functional GABAB receptors. In human SH-SY5Y neuroblastoma cells, CGP7930 and GS39783 induced a time- and concentration-dependent increase in ERK1/2 phosphorylation with potencies similar to those displayed as GABAB PAMs. Conversely, γ-aminobutyric acid and the GABAB receptor agonists (-)baclofen and SKF97541 were completely inactive. CGP7930 and GS39783 enhanced the nuclear localization of phospho-ERK1/2 and CGP7930 promoted the phosphorylation of the transcription factors Elk-1 and CREB. CGP7930-induced ERK1/2 stimulation was insensitive to pertussis toxin, the Gq/11 antagonist YM254890 and the phospholipase C-ß inhibitor U-73122, but was completely blocked by the MEK1/2 inhibitor PD98059. Inhibition of insulin-like growth factor-1, platelet--derived growth factor, phosphoinositide 3-kinase and Akt activities potentiated CGP7930-induced ERK1/2 phosphorylation. CGP7930 enhanced the phosphorylation of myristoylated alanine-rich protein kinase C (PKC) substrate and inhibition of PKC attenuated the ERK1/2 stimulation. Over-expression of N17Ras, a dominant negative mutant of c-Ras, or inhibition of c-Raf by GW5074 partially antagonized CGP7930-induced ERK1/2 activation. CGP7930 enhanced the phosphorylation of transforming growth factor-ß-activated kinase 1 (TAK-1) and TAK-1 inhibition by 5Z-7-oxozeaenol reduced CGP7930-induced ERK1/2 phosphorylation. CGP7930 activated ERK1/2 in CHO-K1 fibroblasts, which lack endogenous GABAB receptors, but not in HEK-293 cells, indicating that the response displayed cell type specificity. These data demonstrate that CGP7930 and GS39783 can trigger ERK1/2 signalling, a critical modulator of mood and drug addiction, independently of an action on GABAB receptors.

LPA1 Mediates Antidepressant-Induced ERK1/2 Signaling and Protection from Oxidative Stress in Glial Cells.

Antidepressants have been shown to affect glial cell functions and intracellular signaling through mechanisms that are still not completely understood. In the present study, we provide evidence that in glial cells the lysophosphatidic acid (LPA) receptor LPA1 mediates antidepressant-induced growth factor receptor transactivation, ERK1/2 signaling, and protection from oxidative stress. Thus, in C6 glioma cells and rat cortical astrocytes, ERK1/2 activation induced by either amitriptyline or mianserin was antagonized by Ki16425 and VPC 12249 (S), which block LPA1 and LPA3 receptors, and by AM966, which selectively blocks LPA1 Cell depletion of LPA1 with siRNA treatment markedly reduced antidepressant- and LPA-induced ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant-induced phosphorylation of the transcription factors CREB and Elk-1. Antidepressants and LPA signaling to ERK1/2 was abrogated by cell treatment with pertussis toxin and by the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases. Both Ki16425 and AM966 suppressed antidepressant-induced phosphorylation of FGF-R. Moreover, blockade of LPA1 or inhibition of FGF-R and PDGF-R activities prevented antidepressant-stimulated Akt and GSK-3ß phosphorylations. Mianserin protected C6 glioma cells and astrocytes from apoptotic cell death induced by H2O2, as indicated by increased cell viability, decreased expression of cleaved caspase 3, reduced cleavage of poly-ADP ribose polymerase and inhibition of DNA fragmentation. The protective effects of mianserin were antagonized by AM966. These data indicate that LPA1 constitutes a novel molecular target of the regulatory actions of tricyclic and tetracyclic antidepressants in glial cells.

Protection from interferon-ß-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1/2 activation.

BACKGROUND AND PURPOSE: Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-ß-induced neuronal apoptosis. EXPERIMENTAL APPROACH: The effects of the ACh receptor agonist carbachol (CCh) on IFN-ß-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using western blots, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and overexpression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-ß-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. KEY RESULTS: In SH-SY5Y cells, CCh inhibited IFN-ß-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 receptors, blocked by Gq/11 antagonist YM254890 and PKC inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by overexpression of ERK2 and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-ß inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, Oxo-M reduced IFN-ß-induced apoptosis; this effect was antagonized by blockade of M1 /M3 receptors and ERK1/2. CONCLUSIONS AND IMPLICATIONS: Stimulation of mAChRs counteracted IFN-ß-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.

Antidepressants activate the lysophosphatidic acid receptor LPA(1) to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts.

Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behavior, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild-type cells. In PathHunter™ assay measuring receptor-ß-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3 stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [(3)H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.

Involvement of store-operated Ca(2+) entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells.

Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca(2+)/calmodulin-dependent protein kinase kinase-ß (CaMKKß)-mediated phosphorylation at Thr172. However, the source of Ca(2+) required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca(2+) entry (SOCE) in AMPK activation by pharmacologically defined M3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca(2+)-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2min. Conversely, in the presence of extracellular Ca(2+) CCh-induced AMPK phosphorylation lasted for at least 180min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50µM) that suppressed CCh-induced intracellular Ca(2+) ([Ca(2+)]i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca(2+)]i plateau and AMPK activation. M3 mAChRs increased glucose uptake and this response required extracellular Ca(2+) and was inhibited by 2-APB, STIM1 knockdown, CaMKKß and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M3 mAChRs and suggests that SOCE is a critical process connecting M3 mAChRs to the control of neuronal energy metabolism.

Interferon-ß counter-regulates its own pro-apoptotic action by activating p38 MAPK signalling in human SH-SY5Y neuroblastoma cells.

Type I interferons (IFNs) induce apoptosis of neuroblastoma cells, but the molecular mechanisms regulating this event have not been completely elucidated. Here, we investigated the role of p38 mitogen activated protein kinase (MAPK) activity, a key regulator of apoptosis and a known modulator of IFN-induced responses in non-neuronal cells. We show that in SH-SY5Y human neuroblastoma cells IFN-ß induced a delayed and sustained increase of p38 MAPK activity through a novel mechanism involving the sequential activation of Janus kinase-signal transducer and activator of transcription-1 signalling, enhanced expression of the NADPH oxidase catalytic subunit gp91(phox), increased reactive oxygen species production and stimulation of the MAPK kinase kinase transforming growth factor-ß-activated kinase 1. Either blockade of p38 MAPK by the second generation inhibitors BIRB0796 and VX745 or siRNA knockdown of p38α MAPK enhanced IFN-ß-induced apoptosis of neuroblastoma cells. Exposure to IFN-ß increased the phosphorylation of the small heat shock protein HSP27 at Ser15, Ser78 and Ser82 with a time course similar to p38 MAPK activation and this response was suppressed by either p38α MAPK depletion or pharmacological inhibition of p38 MAPK and MAPK-activated protein kinase 2 (MK2). Either silencing of HSP27 expression by siRNA or MK2 inhibition potentiated IFN-ß-induced apoptotic death. These results indicate that IFN-ß-induced apoptosis of human SH-SY5Y neuroblastoma cells is associated with a long-lasting up-regulation of p38 MAPK activity, stimulation of MK2 and phosphorylation of the pro-survival protein HSP27. Moreover, the data show that inhibition of p38 MAPK signalling potentiates the anti-neuroblastoma activity of the cytokine, indicating that this pathway mediates a counter-regulatory response.

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen®.

BACKGROUND: Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics. METHODS: Cell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence. RESULTS: The results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®. CONCLUSIONS: BK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients.

Type I interferons up-regulate the expression and signalling of p75 NTR/TrkA receptor complex in differentiated human SH-SY5Y neuroblastoma cells.

Both type I interferons (IFNs) and neurotrophins regulate neuroadaptive responses, but relatively little is known on the interaction between these two classes of regulatory proteins. Here we investigated the effect of IFN-ß on the expression and functional activity of the common neurotrophin receptor p75NTR and the nerve growth factor (NGF) receptor TrkA. In differentiated human SH-SY5Y neuroblastoma cells prolonged exposure to IFN-ß up-regulated p75NTR and TrkA levels, failed to affect the content of sortilin, a p75NTR co-receptor, and, consistent with our previous finding, down-regulated the brain-derived neurotrophic factor receptor TrkB. Quantitative real time RT-PCR indicated that IFN-ß increased p75NTR and TrkA mRNA levels. In control and IFN-ß treated cells proNGF failed to induce c-Jun N-terminal kinase and nuclear factor/kB activation, two p75NTR/sortilin signalling pathways mediating neuronal death. On the other hand, IFN-ß treatment enhanced TrkA autophosphorylation and signalling induced by NGF and proNGF. Knockdown of p75NTR by siRNA reduced TrkA activation by proNGF and a subnanomolar concentration of NGF, whereas co-immunoprecipitation indicated close association of p75NTR and TrkA. Co-treatment with either NGF or proNGF reduced IFN-ß pro-apoptotic and anti-neurotrophic effects. Similarly, in primary mouse hippocampal neurons IFN-ß increased p75NTR and TrkA expression, down-regulated TrkB and enhanced NGF-induced phosphorylation of the pro-survival protein kinase Akt. The data demonstrate that in neuronal cells IFN-ß differentially affects the expression and signalling of neurotrophin receptors and suggest that the up-regulation of the p75NTR/TrkA signalling complex may constitute a novel mechanism by which this cytokine selectively attenuates its pro-apoptotic effect in NGF-responsive cells.

Coincidence signaling of dopamine D1-like and M1 muscarinic receptors in the regulation of cyclic AMP formation and CREB phosphorylation in mouse prefrontal cortex.

In the prefrontal cortex, dopamine D1-like and M1 muscarinic receptors are both involved in the regulation of attentional, cognitive and emotional processes but so far no information has been provided on their functional interaction. In the present study we show that in mouse medial prefrontal cortex, concomitant activation of M1 muscarinic receptors potentiated D1-like receptor-induced cyclic AMP formation through a mechanism involving activation of Gq/11 and the release of G protein ßγ subunits. Immunohistochemical studies indicated that the adenylyl cyclase isoforms AC2 and AC4 are expressed in mouse prefrontal cortex and that they colocalize with D1-like receptors with a greater association for AC4. In primary cultures of frontal cortex neurons, D1-like receptor-induced Ser133 phosphorylation of the transcription factor cyclic AMP-responsive element binding protein (CREB) was potentiated by concurrent stimulation of M1 receptors. Suppression of AC4 expression with small interfering RNA transfection reduced D1 stimulation of cyclic AMP formation and CREB phosphorylation and abolished the M1 potentiation, whereas knockdown of AC2 had no significant effects. These data indicate that in mouse prefrontal cortex Gq/11-coupled M1 receptor and Gs-coupled D1-like receptor inputs converge on AC4 with a consequent enhancement of cyclic AMP formation and signaling to the nucleus.