RESUMO
Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple µ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by µ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFß1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of µ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.
Assuntos
Astrócitos/metabolismo , Morfina/farmacologia , Entorpecentes/farmacologia , Neuritos/metabolismo , Sinapses/metabolismo , Trombospondina 1/biossíntese , Animais , Linhagem Celular Transformada , Proliferação de Células , Córtex Cerebral/metabolismo , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
As embryonic stem cell-derived neural progenitors (NPs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of functional mu opioid receptors (MOR) and kappa opioid receptors (KOR) in mouse embryonic stem cells and NPs. Here, MOR and KOR immunoreactivity was detected in NP-derived oligodendrocytes during three stages of their maturation in vitro. Moreover, we examined the modulation of retinoic acid-induced NP differentiation to astrocytes and neurons by mu, [D-ala(2), mephe(4), gly-ol(5)] enkephalin, or kappa, U69, 593, opioids. Both opioid agonists inhibited NP-derived neurogenesis and astrogenesis via their corresponding receptors as determined by immunocytochemistry. By administering selective inhibitors, we found that opioid inhibition of NP-derived astrogenesis was driven via extracellular-signal regulated kinase (ERK), while the p38 mitogen-activated protein kinase pathway was implicated in opioid attenuation of neurogenesis. In addition, mu and kappa opioids stimulated oligodendrogenesis from NP-derived NG2(+) oligodendrocyte progenitors via both ERK and p38 signaling pathways. Accordingly, both opioids induced ERK phosphorylation in NG2(+) cells. These results indicate that small molecules, such as MOR and KOR agonists may play a modulatory role in NP terminal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Analgésicos Opioides/farmacologia , Animais , Antígenos/metabolismo , Benzenoacetamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Interações Medicamentosas , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Peptídeos/farmacologia , Proteoglicanas/metabolismo , Pirrolidinas/farmacologia , Receptores Opioides kappa/agonistas , Receptores Opioides mu/antagonistas & inibidores , Fatores de Tempo , Tretinoína/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
Acute mu and kappa opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The mu agonist, [D-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas kappa agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant mu opioid receptor that binds CaM poorly or a dominant negative mutant of PKCepsilon were used as a model system to study mu signaling. Evidence was gained to implicate CaM and PKCepsilon in DAMGO stimulation of ERK. DAMGO activation of PKCepsilon and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the mu receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCepsilon. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCzeta, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCzeta. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic mu and kappa opioids.
Assuntos
Astrócitos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase C/química , Receptores Opioides kappa/fisiologia , Receptores Opioides mu/fisiologia , Analgésicos/farmacologia , Analgésicos Opioides/farmacologia , Animais , Astrócitos/citologia , Benzenoacetamidas/farmacologia , Cálcio/metabolismo , Calmodulina/metabolismo , Carbazóis/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Ativação Enzimática , Receptores ErbB/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Indóis , Maleimidas , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Proteína Quinase C-épsilon , Pirrolidinas/farmacologia , Ratos , Receptores Opioides kappa/agonistas , Transdução de Sinais , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. The short-term action of DAMGO resulted in the stimulation of ERK phosphorylation. 4(3-Chlorophenylamino)-6,7-dimethoxyquinazoline (Tyrphostin AG1478), a selective inhibitor of EGFR Tyr kinase activity, blocked EGFR and ERK activation by short-term DAMGO administration, implicating EGFR transactivation in its stimulation of ERK activity. Inhibitors of matrix metalloproteinases attenuated MOR-mediated ERK phosphorylation, suggesting that shedding of EGF-like ligands from the plasma membrane may be involved in the EGFR transactivation process. Prolonged DAMGO exposure induced EGFR internalization/down-regulation, did not activate ERK, and inhibited exogenous EGF-stimulated ERK phosphorylation. MOR-mediated EGFR down-regulation seems to be MAP kinase-dependent, because it was inhibited by the ERK kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126), and tyrphostin AG1478. The kappa opioid agonist (5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) induced Tyr and Ser phosphorylation of EGFR and activation of ERK. However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.
Assuntos
Astrócitos/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Opioides mu/metabolismo , Ativação Transcricional/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Ratos , Receptores Opioides mu/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Mitogenic signaling of G protein-coupled receptors (GPCRs) can proceed via sequential epidermal growth factor receptor (EGFR) transactivation and extracellular signal-regulated kinase (ERK) phosphorylation. Although the mu-opioid receptor (MOR) mediates stimulation of ERK via EGFR transactivation in human embryonic kidney 293 cells, the mechanism of acute MOR signaling to ERK has not been characterized in rat C6 glioma cells that seem to contain little EGFR. Herein, we describe experiments that implicate fibroblast growth factor (FGF) receptor (FGFR) transactivation in the convergence of MOR and growth factor signaling pathways in C6 cells. MOR agonists, endomorphin-1 and morphine, induced a rapid (3-min) increase of ERK phosphorylation that was abolished by MOR antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2. By using selective inhibitors and overexpression of dominant negative mutants, data were obtained to suggest that MOR signaling to ERK is transduced by Gbetagamma and entails Ca2+- and protein kinase C-mediated steps, whereas the FGFR branch of the pathway is Ras-dependent. An intermediary role of FGFR1 transactivation was suggested by MOR- but not kappa-opioid receptor (KOR)-induced FGFR1 tyrosine phosphorylation. A dominant negative mutant of FGFR1 attenuated MOR- but not KOR-induced ERK phosphorylation. Thus, a novel transactivation mechanism entailing secreted endogenous FGF may link the GPCR and growth factor pathways involved in MOR activation of ERK in C6 cells.