RESUMO
BACKGROUND: The G-protein-coupled receptor GPR55 has been implicated in multiple biological activities, which has fuelled interest in its functional targeting. Its controversial pharmacology and often species-dependent regulation have impacted upon the potential translation of preclinical data involving GPR55. RESULTS: With the aim to identify novel GPR55 regulators, we have investigated lysophosphatidylinositol (LPI)-induced GPR55-mediated signal transduction. The expression system for wild-type and mutated GPR55 was HeLa cells silenced for their endogenous receptor by stable expression of a short-hairpin RNA specific for GPR55 5'-UTR, which allowed definition of the requirement of GPR55 Lys80 for LPI-induced MAPK activation and receptor internalisation. In RAW264.7 macrophages, GPR55 pathways were investigated by Gpr55 silencing using small-interfering RNAs, which demonstrated that LPI increased intracellular Ca2+ levels and induced actin filopodium formation through GPR55 activation. Furthermore, the LPI/GPR55 axis was shown to have an active role in osteoclastogenesis of precursor RAW264.7 cells induced by 'receptor-activator of nuclear factor kappa-ß ligand' (RANKL). Indeed, this differentiation into mature osteoclasts was associated with a 14-fold increase in Gpr55 mRNA levels. Moreover, GPR55 silencing and antagonism impaired RANKL-induced transcription of the osteoclastogenesis markers: 'nuclear factor of activated T-cells, cytoplasmic 1', matrix metalloproteinase-9, cathepsin-K, tartrate-resistant acid phosphatase, and the calcitonin receptor, as evaluated by real-time PCR. Phage display was previously used to identify peptides that bind to GPR55. Here, the GPR55-specific peptide-P1 strongly inhibited osteoclast maturation of RAW264.7 macrophages, confirming its activity as a blocker of GPR55-mediated functions. Although osteoclast syncytium formation was not affected by pharmacological regulation of GPR55, osteoclast activity was dependent on GPR55 signalling, as shown with resorption assays on bone slices, where LPI stimulated and GPR55 antagonists inhibited bone erosion. CONCLUSIONS: Our data indicate that GPR55 represents a target for development of novel therapeutic approaches for treatment of pathological conditions caused by osteoclast-exacerbated bone degradation, such as in osteoporosis or during establishment of bone metastases. Video abstract.
Assuntos
Lisofosfolipídeos/metabolismo , Osteogênese , Peptídeos/metabolismo , Receptores de Canabinoides/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Cálcio/metabolismo , Diferenciação Celular , Endocitose , Células HEK293 , Humanos , Ligantes , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Osteoclastos/metabolismo , Pseudópodes/metabolismo , Células RAW 264.7RESUMO
BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.
Assuntos
Movimento Celular , Receptores ErbB/metabolismo , Fosfatos de Inositol/metabolismo , Fosfolipases A2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Sítios de Ligação , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Células NIH 3T3 , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Células RAW 264.7 , Cicatrização , Domínios de Homologia de srcRESUMO
Inflammatory responses are elicited through lipid products of phospholipase A2 activity that acts on the membrane phospholipids, including the phosphoinositides, to form the proinflammatory arachidonic acid and, in parallel, the glycerophosphoinositols. Here, we investigate the role of the glycerophosphoinositol in the inflammatory response. We show that it is part of a negative feedback loop that limits proinflammatory and prothrombotic responses in human monocytes stimulated with lipopolysaccharide. This inhibition is exerted both on the signaling cascade initiated by the lipopolysaccharide with the glycerophosphoinositol-dependent decrease in IκB kinase α/ß, p38, JNK, and Erk1/2 kinase phosphorylation and at the nuclear level with decreased NF-κB translocation and binding to inflammatory gene promoters. In a model of endotoxemia in the mouse, treatment with glycerophosphoinositol reduced TNF-α synthesis, which supports the concept that glycerophosphoinositol inhibits the de novo synthesis of proinflammatory and prothrombotic compounds and might thus have a role as an endogenous mediator in the resolution of inflammation. As indicated, this effect of glycerophosphoinositol can also be exploited in the treatment of manifestations of severe inflammation by exogenous administration of the compound.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Endotoxemia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfatos de Inositol/uso terapêutico , Monócitos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Biomarcadores/sangue , Biomarcadores/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Endotoxemia/imunologia , Endotoxemia/metabolismo , Células HeLa , Humanos , Fosfatos de Inositol/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/sangue , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
The known mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) hydrolyze glycerophosphodiesters. In this study, two novel members of the mammalian GP-PDE family, GDE4 and GDE7, were isolated, and the molecular basis of mammalian GP-PDEs was further explored. The GDE4 and GDE7 sequences are highly homologous and evolutionarily close. GDE4 is expressed in intestinal epithelial cells, spermatids, and macrophages, whereas GDE7 is particularly expressed in gastro-esophageal epithelial cells. Unlike other mammalian GP-PDEs, GDE4 and GDE7 cannot hydrolyze either glycerophosphoinositol or glycerophosphocholine. Unexpectedly, both GDE4 and GDE7 show a lysophospholipase D activity toward lysophosphatidylcholine (lyso-PC). We purified the recombinant GDE4 and GDE7 proteins and show that these enzymes can hydrolyze lyso-PC to produce lysophosphatidic acid (LPA). Further characterization of purified recombinant GDE4 showed that it can also convert lyso-platelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PAF) to alkyl-LPA. These data contribute to our current understanding of mammalian GP-PDEs and of their physiological roles via the control of lyso-PC and lyso-PAF metabolism in gastrointestinal epithelial cells and macrophages.
Assuntos
Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Obesos , Microscopia de Fluorescência , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/genética , Filogenia , Fator de Ativação de Plaquetas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.
Assuntos
Clatrina/metabolismo , Endocitose , Fosfolipases A2 do Grupo IV/fisiologia , Sequência de Aminoácidos , Sinalização do Cálcio , Endossomos/metabolismo , Fosfolipases A2 do Grupo IV/química , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.
Assuntos
Membrana Celular/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Macrófagos/enzimologia , Fagocitose/fisiologia , Fagossomos/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Transformada , Membrana Celular/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Fosfolipases A2 do Grupo IV/genética , Humanos , Macrófagos/citologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Fagocitose/efeitos dos fármacos , Fagossomos/genética , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Pirrolidinas/farmacologia , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMO
Interest in the glycerophosphoinositols has been increasing recently, on the basis of their biological activities. The cellular metabolism of these water-soluble bioactive phosphoinositide metabolites has been clarified, with the identification of the specific enzyme involved in their synthesis, PLA2IVα (phospholipase A2 IVα), and the definition of their phosphodiesterase-based catabolism, and thus inactivation. The functional roles and mechanisms of action of these compounds have been investigated in different cellular contexts. This has led to their definition in the control of various cell functions, such as cell proliferation in the thyroid and actin cytoskeleton organization in fibroblasts and lymphocytes. Roles for the glycerophosphoinositols in immune and inflammatory responses are also being defined. In addition to these physiological functions, the glycerophosphoinositols have potential anti-metastatic activities that should lead to their pharmacological exploitation.
Assuntos
Fosfatos de Inositol/fisiologia , Sistemas do Segundo Mensageiro , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/fisiologia , Fosfolipases A2/metabolismoRESUMO
The organization of intra-Golgi trafficking and the nature of the transport intermediates involved (e.g., vesicles, tubules, or tubular continuities) remain incompletely understood. It was recently shown that successive cisternae in the Golgi stack are interconnected by membrane tubules that form during the arrival of transport carriers from the endoplasmic reticulum. Here, we examine the mechanisms of generation and the function of these tubules. In principle, tubule formation might depend on several protein- and/or lipid-based mechanisms. Among the latter, we have studied the phospholipase A(2) (PLA(2))-mediated generation of wedge-shaped lysolipids, with the resulting local positive membrane curvature. We show that the arrival of cargo at the Golgi complex induces the recruitment of Group IVA Ca(2+)-dependent, cytosolic PLA(2) (cPLA(2)alpha) onto the Golgi complex itself, and that this cPLA(2)alpha is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport. In contrast, silencing of cPLA(2)alpha has no inhibitory effects on peri-Golgi vesicles. These findings identify cPLA(2)alpha as the first component of the machinery that is responsible for the formation of intercisternal tubular continuities and support a role for these continuities in transport through the Golgi complex.
Assuntos
Complexo de Golgi/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Animais , Cálcio/metabolismo , Cães , Complexo de Golgi/ultraestrutura , Fosfolipases A2 do Grupo IV/genética , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Via Secretória , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismoRESUMO
Increasing evidence points to the involvement of group IIA secreted phospholipase A2 (sPLA2-IIA) in pathologies characterized by abnormal osteoclast bone-resorption activity. Here, the role of this moonlighting protein has been deepened in the osteoclastogenesis process driven by the RANKL cytokine in RAW264.7 macrophages and bone-marrow derived precursor cells from BALB/cJ mice. Inhibitors with distinct selectivity toward sPLA2-IIA activities and recombinant sPLA2-IIA (wild-type or catalytically inactive forms, full-length or partial protein sequences) were instrumental to dissect out sPLA2-IIA function, in conjunction with reduction of sPLA2-IIA expression using small-interfering-RNAs and precursor cells from Pla2g2a knock-out mice. The reported data indicate sPLA2-IIA participation in murine osteoclast maturation, control of syncytium formation and resorbing activity, by mechanisms that may be both catalytically dependent and independent. Of note, these studies provide a more complete understanding of the still enigmatic osteoclast multinucleation process, a crucial step for bone-resorbing activity, uncovering the role of sPLA2-IIA interaction with a still unidentified receptor to regulate osteoclast fusion through p38 SAPK activation. This could pave the way for the design of specific inhibitors of sPLA2-IIA binding to interacting partners implicated in osteoclast syncytium formation.
RESUMO
Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Atrofia Muscular/enzimologia , Atrofia Muscular/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Fosfatos de Inositol/metabolismo , Osteoblastos/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proliferação de Células , Humanos , Camundongos , Dados de Sequência Molecular , Osteoblastos/enzimologia , Fosfolipídeos/química , Diester Fosfórico Hidrolases/genética , Homologia de Sequência de AminoácidosRESUMO
The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Lisofosfolipase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Biblioteca Gênica , Humanos , Lisofosfolipase/química , Lisofosfolipase/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/metabolismo , Fosforilação , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Xenopus/crescimento & desenvolvimentoRESUMO
The glycerophosphoinositols are cellular products of phospholipase A(2) and lysolipase activities on the membrane phosphoinositides. Their intracellular concentrations can vary upon oncogenic transformation, cell differentiation and hormonal stimulation. Specific glycerophosphodiester phosphodiesterases are involved in their catabolism, which, as with their formation, is under hormonal regulation. With their mechanisms of action including modulation of adenylyl cyclase, intracellular calcium levels, and Rho-GTPases, the glycerophosphoinositols have diverse effects in multiple cell types: induction of cell proliferation in thyroid cells; modulation of actin cytoskeleton organisation in fibroblasts; and reduction of the invasive potential of tumour cell lines. More recent investigations include their effects in inflammatory and immune responses. Indeed, the glycerophosphoinositols enhance cytokine-dependent chemotaxis in T-lymphocytes induced by SDF-1alpha-receptor activation, indicating roles for these compounds as modulators of T-cell signalling and T-cell responses.
Assuntos
Células/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/fisiologia , Animais , Fenômenos Fisiológicos Celulares , Humanos , Lipídeos/química , Lipídeos/fisiologia , Modelos Biológicos , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/fisiologia , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
Recent insights have indicated an active role of the complex complement system not only in immunity, but also in bone remodeling. Evidence from knockout mice and observations from skeletal diseases have drawn attention to the C5a/C5aR axis of the complement cascade in the modulation of osteoclast functions and as potential therapeutic targets for treatment of bone pathologies. With the aim to identify novel C5aR regulators, a medicinal chemistry program was initiated, driven by structural information on a minor pocket of C5aR that has been proposed to be a key motif for C5aR intracellular activation. The impact of the peptidomimetic orthosteric C5aR antagonist (PMX-53), of two newly synthesized allosteric C5aR antagonists (DF2593A, DF3016A), and of C5aR down-regulation by specific siRNAs, were examined for regulation of osteoclastogenesis, using a well-validated in-vitro model starting from RAW264.7 precursor cells. Both pharmacological and molecular approaches reduced osteoclast maturation of RAW264.7 cells induced by receptor-activator of nuclear factor kappa-B ligand (RANKL), which limited the transcription of several differentiation markers evaluated by real-time PCR, including nuclear factor of activated T-cell 1, matrix metalloproteinase-9, cathepsin-K, and tartrate-resistant acid phosphatase. These treatments were ineffective on the subsequent step of osteoclast syncytium formation, apparently as a consequence of reduction of C5aR mRNA levels in the course of osteoclastogenesis, as monitored by real-time PCR. Among the C5aR antagonists analyzed, DF3016A inhibited osteoclast degradation activity through inhibition of C5aR signal transduction and transcription. These data confirm the preclinical relevance of this novel therapeutic candidate.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Tetrazóis/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Células RAW 264.7RESUMO
The glycerophosphoinositols are diffusible phosphoinositide metabolites reported to modulate actin dynamics and tumour cell spreading. In particular, the membrane permeant glycerophosphoinositol 4-phosphate (GroPIns4P) has been shown to act at the level of the small GTPase Rac1, to induce the rapid formation of membrane ruffles. Here, we have investigated the signalling cascade involved in this process, and show that it is initiated by the activation of Src kinase. In NIH3T3 cells, exogenous addition of GroPIns4P induces activation and translocation of Rac1 and its exchange factor TIAM1 to the plasma membrane; in addition, in in-vitro assays, GroPIns4P favours the formation of a protein complex that includes Rac1 and TIAM1. Neither of these processes involves direct actions of GroPIns4P on these proteins. Thus, through the use of specific inhibitors of tyrosine kinases and phospholipase C (and by direct evaluation of kinase activities and inositol 1,4,5-trisphosphate production), we show that GroPIns4P activates Src, and as a consequence, phospholipase Cgamma and Ca(2+)/calmodulin kinase II, the last of which directly phosphorylates TIAM1 and leads to TIAM1/Rac1-dependent ruffle formation.
Assuntos
Actinas/metabolismo , Fosfatos de Inositol/farmacologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Células NIH 3T3 , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
Glycerophosphoinositols (GPIs) are water-soluble phosphoinosite metabolites produced by all cell types, whose levels increase in response to a variety of extracellular stimuli, and are particularly high in Ras-transformed cells. GPIs are released to the extracellular space, wherefrom they can be taken up by other cells through a specific transporter. Exogenous GPIs affect a plethora of cellular functions. Among these compounds the most active is GroPIns4P, which affects cAMP levels and PKA-dependent functions through the inhibition of heterotrimeric Gs proteins. GroPIns4P has also recently been found to promote actin cytoskeleton reorganization by inducing Rho and Rac activation through an as yet unidentified mechanism. Here we have assessed the potential effects of GroPIns4P on T-cells. We found that GroPIns4P enhances CXCR4-dependent chemotaxis. This activity results from the capacity of GroPIns4P to activate the Rho GTPase exchange factor, Vav, through an Lck-dependent pathway which also results in activation of the stress kinases JNK and p38. GroPIns4P was also found to activate with a delayed kinetics the Lck-dependent activation of ZAP-70, Shc and Erk1/2. The activities of GroPIns4P were found to be dependent on its capacity to inhibit cAMP production and PKA activation. Collectively, the data provide the first evidence of a role of glycerophosphoinositols as modulators of T-cell signaling and establish a mechanistic basis for the effects of this phosphoinositide derivative on F-actin dynamics.
Assuntos
Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Fosfatos de Inositol/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CXCL12 , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with synaptojanin 1 [1,2]. Neurons of synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type synaptojanin 2, but not of wild-type synaptojanin 1 or mutant synaptojanin 2 that is deficient in 5'-phosphatase activity. In addition, electron-microscopic analysis shows that synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that synaptojanin 2 is functionally distinct from synaptojanin 1.
Assuntos
Vesículas Revestidas por Clatrina/ultraestrutura , Endocitose/fisiologia , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Imunofluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Microscopia Eletrônica , RNA Interferente Pequeno/metabolismoRESUMO
The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.
Assuntos
Células Epiteliais/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipases A/metabolismo , Animais , Células CHO , Diferenciação Celular , Linhagem Celular , Cricetinae , Cricetulus , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Marcadores Genéticos/fisiologia , Fosfolipases A2 do Grupo IV , Fosfolipases A2 , RNA Mensageiro/metabolismo , Ratos , Receptores Purinérgicos/metabolismo , Glândula Tireoide/citologiaRESUMO
G-protein-coupled-receptor kinase 2 (GRK2) plays a key role in the modulation of G-protein-coupled-receptor (GPCR) signaling by both phosphorylating agonist-occupied GPCRs and by directly binding to activated Galphaq subunits, inhibiting downstream effectors activation. The GRK2/Galphaq interaction involves the N-terminal region of the kinase that displays homology to regulators of G-protein signaling (RGS) proteins. We have previously reported that upon GPCR stimulation, GRK2 can be phosphorylated by c-Src on tyrosine residues that are present in the RGS-homology (RH) region of this kinase. Here, we demonstrate that c-Src kinase activity increases the interaction between GRK2 and Galphaq. Tyrosine phosphorylation of GRK2 appears to be critically involved in the modulation of this interaction since the stimulatory effect of c-Src is not observed with a GRK2 mutant with impaired tyrosine phosphorylation (GRK2 Y13,86,92F), whereas a mutant that mimics GRK2 tyrosine phosphorylation in these residues displays an increased interaction with Galphaq. As evidence for a physiological role of this modulatory mechanism, activation of the muscarinic receptor M1, a Galphaq-coupled receptor, promotes an increase in GRK2/Galphaq co-immunoprecipitation that parallels the enhanced GRK2 phosphorylation on tyrosine residues. Moreover, c-Src activation enhances inhibition of the Galphaq/phospholipase Cbeta signaling pathway in intact cells, in a GRK2-tyrosine-phosphorylation-dependent manner. Our results suggest a feedback mechanism by which phosphorylation of GRK2 by c-Src increases both GRK2 kinase activity towards GPCRs and its specific interaction with Galphaq subunits, leading to a more rapid switch off of Galphaq-mediated signaling.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Quinases de Receptores Adrenérgicos beta/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular , Quinase 2 de Receptor Acoplado a Proteína G , Humanos , Fosforilação , Ligação Proteica/fisiologia , Quinases da Família srcRESUMO
Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.