RESUMO
Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.
Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais/fisiologiaRESUMO
AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.
Assuntos
Bestrofinas/metabolismo , Ácido Glutâmico/metabolismo , Peptídeos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Peptídeos/administração & dosagem , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/citologiaRESUMO
PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Receptor PAR-1/metabolismo , Retina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Humanos , Retina/citologiaRESUMO
Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.
Assuntos
Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/citologia , Trocador de Sódio e Cálcio/metabolismo , Trombina/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Forma Celular/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Fragmentos de Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos Long-Evans , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trítio/metabolismoRESUMO
The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by Gq11α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of µ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel µ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.
RESUMO
Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK)-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis.
Assuntos
Movimento Celular , Paxilina/metabolismo , Animais , Adesões Focais/metabolismo , Humanos , Patologia Molecular , Fosforilação , Transdução de SinaisRESUMO
Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function.
Assuntos
Células Ependimogliais/metabolismo , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Receptores de Glutamato/metabolismo , Regulação para Cima/fisiologia , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Células Cultivadas , Células Ependimogliais/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Neuroglia/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The breakdown of the blood-retina barrier exposes retinal pigment epithelium (RPE) to serum components, thrombin among them. In addition to coagulation, thrombin acting through Protease-Activated Receptors (PARs 1-4) participates in a number of processes including cell proliferation, transformation, and migration. The purpose of this study was to identify interacting signaling pathways by which the activation of PAR1 by thrombin triggers cyclin D1 gene (Ccnd1) expression and the proliferation of RPE cells, characteristic of proliferative vitreoretinopathy (PVR). Our results demonstrate that thrombin induces the expression of the c-fos gene (c-fos), the activation of the (fos/jun) AP-1 site and the expression of Ccnd1, in precise correlation with the activation of CREB. Although the expression of both, c-fos and Ccnd1 requires the activation of conventional PKC isoforms and PI3K, downstream signaling from PI3K differs for both genes. Whereas the expression of c-fos requires PI3K-induced PDK1/Akt activity, that of Ccnd1 is mediated by PDK1-independent PKCζ signaling. Additionally, CREB activation may contribute to the induction of Ccnd1 expression through binding to the Ca/CRE element in the Ccnd1 gene promoter. Since cyclin D1 is a key regulator of cell cycle G1/S phase progression essential for proliferation, these findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.
Assuntos
Ciclina D1/genética , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Vitreorretinopatia Proliferativa/genética , Barreira Hematorretiniana/efeitos dos fármacos , Western Blotting , Proliferação de Células , Células Cultivadas , Ciclina D1/biossíntese , Hemostáticos/farmacologia , Humanos , Reação em Cadeia da Polimerase , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Trombina/farmacologia , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-ζ/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Trombina/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , RNA Interferente Pequeno , Proteína Companheira de mTOR Insensível à Rapamicina , Ratos , Proteína Regulatória Associada a mTOR , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
The proliferation, directional migration to the vitreous and epithelial-mesenchymal transition (EMT) of quiescent, differentiated retinal pigment epithelium (RPE) cells is a major feature in the development of proliferative vitreoretinopathy (PVR) following exposure of the immuno-privileged eye niche to serum components, thrombin among them. We have previously documented thrombin induction of RPE cell proliferation and migration. We here analyzed the effect of thrombin on the E/N cadherin switch, a hallmark of EMT. Results show that thrombin induces the specific repression of epithelial E-cadherin gene transcription, alongside with the up-regulation of mesenchymal N-cadherin protein in RPE cells. We demonstrate, for the first time, that thrombin induces E-cadherin repression by stimulating snail-2 (SLUG) transcription factor expression, and the concomitant up-regulation of N-cadherin through the transcription-independent increase in protein translation promoted by PI3K/PKC-ζ/mTOR signaling. Our present findings suggest that the activation of protease-activated receptor-1 (PAR-1) by thrombin induces EMT of RPE cells, further supporting a central role for thrombin in PVR pathogenesis.
Assuntos
Caderinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Trombina/farmacologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Caderinas/antagonistas & inibidores , Caderinas/genética , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Receptor PAR-1/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail , Serina-Treonina Quinases TOR , Trombina/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vitreorretinopatia Proliferativa/etiologiaRESUMO
Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.
Assuntos
Hemostáticos/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fibras de Estresse/metabolismo , Trombina/farmacologia , Actinas/metabolismo , Animais , Barreira Hematorretiniana , Western Blotting , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Proteínas Musculares , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The retinal pigment epithelium (RPE) plays an essential role in the function of the neural retina and the maintenance of vision. Most of the functions displayed by RPE require a dynamic organization of the acto-myosin cytoskeleton. Myosin II, a main cytoskeletal component in muscle and non-muscle cells, is directly involved in force generation required for organelle movement, selective molecule transport within cell compartments, exocytosis, endocytosis, phagocytosis, and cell division, among others. Contractile processes are triggered by the phosphorylation of myosin II light chains (MLCs), which promotes actin-myosin interaction and the assembly of contractile fibers. Considerable evidence indicates that non-muscle myosin II activation is critically involved in various pathological states, increasing the interest in studying the signaling pathways controlling MLC phosphorylation. Particularly, recent findings suggest a role for non-muscle myosin II-induced contraction in RPE cell transformation involved in the establishment of numerous retinal diseases. This review summarizes the current knowledge regarding myosin function in RPE cells, as well as the signaling networks leading to MLC phosphorylation under pathological conditions. Understanding the molecular mechanisms underlying RPE dysfunction would improve the development of new therapies for the treatment or prevention of different ocular disorders leading to blindness.
Assuntos
Células Epiteliais/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Células Epiteliais/fisiologia , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/fisiologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/fisiologia , Fosforilação/genética , Epitélio Pigmentado da Retina/fisiologiaRESUMO
The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-ß inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC-ß-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.
Assuntos
Actinas/metabolismo , Barreira Hematorretiniana/metabolismo , Cadeias Leves de Miosina/metabolismo , Epitélio Pigmentado da Retina , Fibras de Estresse/metabolismo , Trombina/farmacologia , Quinases Associadas a rho/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C beta/metabolismo , Fosforilação , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The retinal pigment epithelium (RPE) plays an essential role in the maintenance and normal functioning of the neural retina. Alterations in RPE function are involved in several ocular pathologies involving the breakdown of the blood-retina barrier (BRB), which exposes RPE to serum components, thrombin among them. Our previous work has shown that thrombin stimulates the proliferation of RPE cells. We here analyzed the molecular pathways leading to this outcome, in order to support thrombin involvement in proliferative vitreoretinopathy (PVR), a major cause of retinal surgery failure. We demonstrated that thrombin activation of PAR-1 promotes cyclin D1 expression at the transcriptional level by stimulating c-Fos expression, mediated by PI3K, MAPK ERK1/2, and conventional PKC activity. Our results show that ERK activation is necessary but not sufficient for the induction of cyclin D1 expression and proliferation, since the inhibition of PI3K or cPKC prevents this outcome. Analysis of thrombin-activated PAR-1 downstream effectors demonstrated that c-Fos expression by the sustained activation of ERK and c-fos transcription triggers the expression and nuclear translocation of cyclin D1, a key regulator of cell cycle G1/S phase progression leading to proliferation. Evidence here provided contributes to the understanding of the mechanisms involved in proliferative eye diseases and enhances the possibility of controlling pathologies such as proliferative PVR, which eventually lead to blindness.
Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Células Epiteliais/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trombina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C beta/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Vitreorretinopatia Proliferativa/patologiaRESUMO
Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hemostáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trombina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Hemostáticos/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Isoenzimas , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Long-Evans , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Trombina/metabolismo , Vitreorretinopatia Proliferativa/metabolismoRESUMO
The N-methyl-D-aspartate type of glutamate receptor (NMDAR) plays a major role in the vertebrate retina. Expression of NR1 splice-variants and NR2 subunits in the retina differs from that in the brain, suggesting a tissue-specific heteromeric assembly of NMDARs. We previously demonstrated that serum alters retinal glutamate receptor properties. In order to relate this effect to NMDAR subunit composition, we here studied the effect of serum on the expression of NMDAR subunits and splice-variants in chick retinal neurons in primary culture. Our results show that mRNA and protein expression of NR1 alternative splice-variants and NR2 subunits are differentially modified by glutamate contained in serum. Such alteration suggests that NMDAR structure is reversed to embryonic heteromeric composition, through the control of subunit availability. The present findings could be relevant for the understanding of the lack of effect in the retina, of drugs which have been shown to protect cortical neurons from glutamate-induced excitotoxicity in those pathological or clinical conditions in which the retina is exposed to serum.
Assuntos
Neurônios/metabolismo , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/citologia , Soro/metabolismo , Transcrição Gênica , Animais , Embrião de Galinha , Antagonistas de Aminoácidos Excitatórios/metabolismo , Genisteína/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Neurônios/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Subunidades Proteicas/genética , Quinoxalinas/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Retina/metabolismoRESUMO
Retinal pigment epithelial (RPE) cells are the main cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR). As a result from retinal detachment or surgical procedures, RPE comes in contact with glutamate from serum, glial release and the injured retina. The purpose of this study was to explore a possible role for glutamate in the development of PVR, mediated by the receptor-stimulated activation of the ERK1/2 MAPK pathway, the alteration of cell proliferation and the transdifferentiation of RPE cells, using rat RPE cells in culture as a model system. We demonstrated the expression in these cells of Group I metabotropic-and ionotropic AMPA/KA and NMDA glutamate receptors (GluRs), predominantly of the NMDA subtype, which are targeted to the membrane, and exhibit pharmacological and biochemical characteristics equivalent to those previously established in brain tissue. Proliferation was measured by MTS-reduction colorimetric assay, and actin cytoskeleton dynamics was visualized by immunoflurescence using alpha-sma specific antibodies. Activation of metabotropic, AMPA and NMDA receptors by glutamate induced the time-and dose-dependent phosphorylation of ERK1/2, assessed by Western blot analysis, in parallel to a significant increase in cell proliferation and a decrease in alpha-sma expression and its recruitment into stress fibers. These effects were all prevented by the inhibition of MEK. Hence, results suggest that glutamate could be involved in the generation of PVR, through a GluR-mediated increase in proliferation and phenotypic transformation, cause-effect related to the activation of ERK1/2.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Epitélio Pigmentado Ocular/citologia , Receptores de Glutamato/fisiologia , Vitreorretinopatia Proliferativa/patologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática/fisiologia , Ácido Glutâmico/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia Confocal , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/enzimologia , Epitélio Pigmentado Ocular/patologia , Ratos , Ratos Long-Evans , Vitreorretinopatia Proliferativa/enzimologia , Vitreorretinopatia Proliferativa/metabolismoRESUMO
The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proliferação de Células/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Epitélio Pigmentado Ocular/citologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases , Receptores de Glutamato/metabolismo , Receptores de Glutamato/fisiologia , Transdução de SinaisRESUMO
D-serine is an N-methyl-D-aspartate (NMDA) receptor coagonist predominantly produced by glial cells in the brain and the retina. Whereas a role for D-serine as a modulator of NMDA receptors in neurons has been suggested, its function in glial cells has not been analyzed. We here show that D-serine modulates gene expression in Müller glial cells from the retina through the induction of transcription factor CREB phosphorylation and the expression of the immediate-early gene c-fos. Pharmacological analysis indicates that D-serine effect involves NMDA receptor activation. Comparison of the effect of D-serine in Müller cells, hippocampal astrocytes and hippocampal neurons suggests that D-serine could function as a retinal NMDA receptor coagonist activating functionally relevant transcription factor pathways in glial cells.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/genética , Neuroglia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Serina/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/agonistas , Retina/citologia , Serina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Las bases celulares y moleculares de los procesos de envejecimiento y de la plasticidad en el sistema nervioso (SN) no se han esclarecido totalmente. Este trabajo tiene como finalidad explorar el problema desde un punto de vista embriológico, partiendo de la idea de que en el SN hay diferentes microambientes, predeterminados por la ontogenia del organismo, que tienen repercusión directa sobre su funcionamiento.En este contexto se analizan algunos de los cambios en la composición heteromérica de los receptores de glutamato tipo NMDA que tienen lugar durante el desarrollo embrionario, y sus consecuencias funcionales, comparándolos con los cambios equivalentes, identificados en el tejido nervioso diferenciado, como consecuencia de fenómenos plásticos. Finalmente, aplicando los resultados de este análisis se propondrá una hipótesis que relaciona los cambios mencionados con el proceso de envejecimiento y la enfermedad de Alzheimer.