RESUMO
Many G protein-coupled receptors (GPCRs), including serotonin (5-HT) receptors promote the activity of receptor tyrosine kinases (RTKs) via intracellular signaling pathways in a process termed transactivation. Although transactivation pathways are commonly initiated by a GPCR, a recent report demonstrated that serotonin-selective reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of the platelet-derived growth factor (PDGF) type ß receptor. We show that a 45 min pretreatment of SH-SY5Y cells with the SSRI fluoxetine indeed blocked 5-HT-induced transactivation of the PDGFß receptor. However, upon further examination, we discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFß receptor via 5-HT2 receptor activation. After 45min, the increase in PDGFß receptor phosphorylation induced by fluoxetine had returned to baseline, but a subsequent transactivating stimulus (5-HT) failed to "re-transactivate" the PDGFß receptor. We further demonstrate that 45min, but not 3h, 5-HT pretreatment blocks dopamine-induced PDGFß receptor transactivation. This did not involve changes in PDGF receptor function, since ligand (PDGF)-induced PDGFß receptor activation was not inhibited by 5-HT pretreatment. To our knowledge this is the first demonstration of the heterologous desensitization of an RTK transactivation pathway and reveals a previously unknown short-term "blackout" period where no additional transactivation signaling is possible.
Assuntos
Fluoxetina/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/agonistas , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors regulates growth factor receptor expression, including the expression of platelet-derived growth factor (PDGF) ß receptors. Direct activation of PDGFß receptors in primary hippocampal and cortical neurons inhibits NMDA receptor activity and attenuates NMDA receptor-induced neurotoxicity. Our objective was to investigate whether the 5-HT7 receptor-induced increase in PDGFß receptor expression would be similarly neuroprotective. We demonstrate that 5-HT7 receptor agonist treatment in primary hippocampal neurons also increases the expression of phospholipase C (PLC) γ, a downstream effector of PDGFß receptors associated with the inhibition of NMDA receptor activity. To determine if the up-regulation of PDGFß receptors is neuroprotective, primary hippocampal neurons were incubated with the 5-HT7 receptor agonist, LP 12, for 24 h. Indeed, LP 12 treatment prevented NMDA-induced neurotoxicity and this effect was dependent on PDGFß receptor kinase activity. Treatment of primary neurons with LP 12 also differentially altered NMDA receptor subunit expression, reducing the expression of NR1 and NR2B, but not NR2A. These findings demonstrate the potential for providing growth factor receptor-dependent neuroprotective effects using small-molecule ligands of G protein-coupled receptors.
Assuntos
N-Metilaspartato/fisiologia , Neurônios/citologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Serotonina/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Camundongos , N-Metilaspartato/toxicidade , Neurônios/metabolismo , Fosforilação , Piperazinas/farmacologia , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Agonistas do Receptor de Serotonina/farmacologia , Fosfolipases Tipo C/metabolismo , Regulação para CimaRESUMO
BACKGROUND: PDGFß receptors and their ligand, PDGF-BB, are upregulated in vivo after neuronal insults such as ischemia. When applied exogenously, PDGF-BB is neuroprotective against excitotoxicity and HIV proteins. OBJECTIVE: Given this growth factor's neuroprotective ability, we sought to determine if PDGF-BB would be neuroprotective against amyloid-ß (1-42), one of the pathological agents associated with Alzheimer's disease (AD). METHODS AND RESULTS: In both primary hippocampal neurons and the human-derived neuroblastoma cell line, SH-SY5Y, amyloid-ß treatment for 24 h decreased surviving cell number in a concentrationdependent manner. Pretreatment with PDGF-BB failed to provide any neuroprotection against amyloid-ß in primary neurons and only very limited protective effects in SH-SY5Y cells. In addition to its neuroprotective action, PDGF promotes cell growth and division in several systems, and the application of PDGFBB alone to serum-starved SH-SY5Y cells resulted in an increase in cell number. Amyloid-ß attenuated the mitogenic effects of PDGF-BB, inhibited PDGF-BB-induced PDGFß receptor phosphorylation, and attenuated the ability of PDGF-BB to protect neurons against NMDA-induced excitotoxicity. Despite the ability of amyloid-ß to inhibit PDGFß receptor activation, immunoprecipitation experiments failed to detect a physical interaction between amyloid-ß and PDGF-BB or the PDGFß receptor. However, G protein-coupled receptor transactivation of the PDGFß receptor (an exclusively intracellular signaling pathway) remained unaffected by the presence of amyloid-ß. CONCLUSIONS: As the PDGF system is upregulated upon neuronal damage, the ability of amyloid-ß to inhibit this endogenous neuroprotective system should be further investigated in the context of AD pathophysiology.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Becaplermina/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Neuroproteção/fisiologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Serotonina/metabolismoRESUMO
High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type ß receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H2O2 induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H2O2 failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFß receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFß receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFß and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFß and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor trkB/metabolismo , Serotonina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , NADPH Oxidases/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
In the absence of ligand, certain growth factor receptors can be activated via G-protein coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate platelet-derived growth factor (PDGF) ß receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here we show that 5-HT can transiently increase the phosphorylation of PDGFß receptors through 5-HT(1A) receptors in a time- and dose-dependent manner in SH-SY5Y neuroblastoma cells. 5-HT also transactivates PDGFß receptors in primary cortical neurons. This transactivation pathway is pertussis-toxin sensitive and Src tyrosine kinase-dependent. This pathway is also dependent on phospholipase C activity and intracellular calcium signaling. Several studies involving PDGFß receptor transactivation by GPCRs have also demonstrated a PDGFß receptor-dependent increase in the phosphorylation of ERK1/2. Yet in SH-SY5Y cells, 5-HT treatment causes a PDGFß receptor-independent increase in ERK1/2 phosphorylation. This crosstalk between 5-HT and PDGFß receptors identifies a potentially important signaling link between the serotonergic system and growth factor signaling in neurons.
Assuntos
Neurônios/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Animais , Becaplermina , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Serotonina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) ß receptors which can protect neurons against NMDA-induced neurotoxicity. RESULTS: In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered. CONCLUSIONS: In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.
Assuntos
Hipocampo/citologia , Ativação do Canal Iônico/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Serotonina/metabolismo , Amidas/farmacologia , Animais , Modelos Biológicos , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Piperazinas/farmacologia , Ratos Wistar , Serotonina/análogos & derivados , Serotonina/farmacologiaRESUMO
Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)ß receptors. The increased PDGFß receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFß receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.