RESUMEN
Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, ãincluding Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.
Asunto(s)
Estudio de Asociación del Genoma Completo , Hierro , Hierro/metabolismo , Metilación de ADN/genética , Epigénesis Genética , Adipocitos/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismoRESUMEN
The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF.
Asunto(s)
Ácidos/metabolismo , Embrión de Mamíferos/efectos de los fármacos , Toxina del Pertussis/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Canales Iónicos Sensibles al Ácido/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Concentración de Iones de Hidrógeno , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPV/metabolismoRESUMEN
Although blood pH is maintained in a narrow range of around pH 7.4 in living organisms, inflammatory loci are characterized by acidic conditions. Mast cells tend to reside close to the surface of the body in areas such as the mucosa and skin where they may be exposed to exogenous acids, and they play an important role in immune responses. However, little is known about the effects of extracellular acidification on the functions of mast cell. Here, we found that extracellular acidification increased the dinitrophenyl-conjugated human serum albumin (DNP-HSA)-induced production of interleukin (IL)-6 and IL-13 in MC/9 cells or bone marrow-derived mouse mast cells sensitized with anti-DNP IgE. Extracellular acidification also inhibited migration of MC/9 cells toward DNP-HSA. In addition, acidic pH stimulated antigen-induced activation of p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). These findings suggest that extracellular acidification augmented antigen/IgE-induced and FcεRI-mediated production of IL-6 and IL-13 in mast cells, and that this was associated with the enhancement of p38 MAPK and Akt activation.
Asunto(s)
Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Dinitrofenoles/farmacología , Concentración de Iones de Hidrógeno , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Mastocitos/química , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Albúmina Sérica/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Pulmón/patología , Lesión Pulmonar Aguda/genética , Animales , Quimiocinas/inmunología , Femenino , Eliminación de Gen , Lipopolisacáridos/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/patología , Protones , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Regulación hacia ArribaRESUMEN
Interleukin-1ß (IL-1ß) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL-1ß production, especially focusing on TDAG8, a major proton-sensing G-protein-coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide -induced IL-1ß production, which was associated with the inhibition of IL-1ß cytoplasmic precursor and mRNA expression. The IL-1ß mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8-deficient mice compared with those from wild-type mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH-induced IL-1ß mRNA expression. The acidic pH-induced inhibitory IL-1ß responses were accompanied by the inhibition of extracellular signal-related kinase and c-Jun N-terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide-induced IL-1ß production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signal-related kinase and c-Jun N-terminal kinase activities, in mouse microglia.
Asunto(s)
AMP Cíclico/fisiología , Líquido Extracelular/química , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/biosíntesis , Microglía/metabolismo , Sistemas de Mensajero Secundario/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Concentración de Iones de Hidrógeno , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Poli I-C/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/fisiología , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. Here, we show that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. Our work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body.
Asunto(s)
Drosophila , Proteostasis , Animales , Cuerpo Adiposo , Proteína p53 Supresora de Tumor , Muerte Celular , LarvaRESUMEN
Pancreatic cancer is highly metastatic and has a poor prognosis. However, there is no established treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in effluents of cancers and involved in migration and proliferation in a variety of cancer cells, including pancreatic cancer cells, in vitro. In the current study, we examined whether an orally active LPA antagonist is effective for pancreatic cancer tumorigenesis and metastasis in vivo. Oral administration of Ki16198, which is effective for LPA(1) and LPA(3), into YAPC-PD pancreatic cancer cell-inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA-induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA-induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells.
Asunto(s)
Isoxazoles/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Propionatos/farmacología , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Animales , Ascitis/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Isoxazoles/administración & dosificación , Isoxazoles/uso terapéutico , Lisofosfolípidos/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Peritoneales/patología , Neoplasias Peritoneales/prevención & control , Neoplasias Peritoneales/secundario , Propionatos/administración & dosificación , Propionatos/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Glucocorticoides/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Concentración de Iones de Hidrógeno , Macrófagos Peritoneales/metabolismo , Ratones , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Extracellular acidification inhibited LPS-induced TNF-alpha protein production, which was associated with an inhibition of TNF-alpha mRNA expression, in mouse peritoneal macrophages. The LPS-induced cytokine production was also inhibited by G(s) protein-coupled receptor agonists prostaglandin E(1) and isoproterenol. Among OGR1 family proton-sensing GTP-binding regulatory protein-coupled receptors, TDAG8, OGR1, and G2A are expressed in the cells. The inhibitory action by acidic pH on TNF-alpha production was significantly attenuated in macrophages from TDAG8(Tp/Tp) mice but not in those from OGR1(geo/geo) mice. Moreover, small interfering RNA specific to TDAG8, but not to G2A, clearly attenuated the acidification-induced inhibition of TNF-alpha production. On the other hand, the down-regulation or deficiency of TDAG8 hardly affected prostaglandin E(1)- or isoproterenol-induced actions. LPS-induced IL-6 production was also inhibited by extracellular acidification in a manner that was sensitive to TDAG8 expression. The acidic pH-induced inhibitory action on the cytokine production was significantly reversed either by a small interfering RNA specific to G(s) proteins or by a protein kinase A (PKA)-specific inhibitor H89. Indeed, a PKA-specific cAMP derivative inhibited LPS-induced cytokine production. Moreover, acidification induced cAMP accumulation in a TDAG8-specific way. We conclude that TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production through the G(s) protein/cAMP/PKA signaling pathway in mouse macrophages.
Asunto(s)
Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Espacio Extracelular/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Protones , Receptores Acoplados a Proteínas G/fisiología , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Espacio Extracelular/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Concentración de Iones de Hidrógeno , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.
Asunto(s)
Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análisis de Varianza , Western Blotting , Células Cultivadas , AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Epoprostenol/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Músculo Liso Vascular/citología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the G(s)-protein/cAMP, G(12/13)-protein/Rho, and G(q)-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC(50)) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins.
Asunto(s)
Histidina/genética , Mutación Puntual , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Línea Celular , Humanos , ProtonesRESUMEN
While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E(2) production, in RA synovial cells. The synovial fluid-induced actions were inhibited by G(i/o) protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1alpha- or IL-1beta-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA(1) receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Ciclooxigenasa 2/biosíntesis , Fibroblastos/metabolismo , Lisofosfolípidos/fisiología , Líquido Sinovial/enzimología , Líquido Sinovial/inmunología , Adyuvantes Inmunológicos/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Línea Celular Tumoral , Citocinas/fisiología , Dinoprostona/biosíntesis , Dinoprostona/fisiología , Inducción Enzimática/inmunología , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Lisofosfolípidos/metabolismo , Propionatos/farmacología , Propionatos/uso terapéutico , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Líquido Sinovial/citologíaRESUMEN
Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.
Asunto(s)
Lesiones Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Sustancias Protectoras/metabolismo , Animales , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Protones , Receptores Acoplados a Proteínas G/metabolismo , Reperfusión/métodos , Transducción de Señal/fisiologíaRESUMEN
Malignant ascites from pancreatic cancer patients has been reported to stimulate migration of pancreatic cancer cells through lysophosphatidic acid (LPA) and LPA(1) receptors. Indeed, ascites- and LPA-induced migration was inhibited by Ki16425, an LPA(1) and LPA(3) antagonist, in Panc-1 cells. Unexpectedly, however, in the presence of Ki16425, ascites and LPA inhibited cell migration in response to epidermal growth factor (EGF). The inhibitory migratory response to ascites and LPA was also observed in the cells treated with pertussis toxin (PTX), a G(i) protein inhibitor, and attenuated by a small interfering RNA (siRNA) specific to the LPA(2) receptor. The inhibitory LPA action was reversed by the regulators of G-protein signaling domain of p115RhoGEF, dominant-negative RhoA or C3 toxin. Indeed, LPA activated RhoA, which was attenuated by the siRNA against the LPA(2) receptor. Moreover, LP-105, an LPA(2) agonist, also inhibited EGF-induced migration in the PTX-treated cells. A similar inhibitory migration response through LPA(2) receptors was also observed in YAPC-PD, BxPC-3, CFPAC-1 and PK-1 pancreatic cancer cell lines. LPA also inhibited the invasion of Panc-1 cells in the PTX-treated cells in the in vitro Matrigel invasion assay. We conclude that LPA(2) receptors are coupled to the G(12/13) protein/Rho-signaling pathway, leading to the inhibition of EGF-induced migration and invasion of pancreatic cancer cells.
Asunto(s)
Ascitis/metabolismo , Movimiento Celular/efectos de los fármacos , Lisofosfolípidos/farmacología , Neoplasias Pancreáticas/metabolismo , Receptores del Ácido Lisofosfatídico/fisiología , Ascitis/patología , Línea Celular Tumoral , Colágeno , Combinación de Medicamentos , Factor de Crecimiento Epidérmico/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Humanos , Isoxazoles/farmacología , Laminina , Invasividad Neoplásica , Neoplasias Pancreáticas/patología , Toxina del Pertussis/farmacología , Propionatos/farmacología , Proteoglicanos , ARN Interferente Pequeño/genética , Receptores del Ácido Lisofosfatídico/agonistas , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O(2)(-)) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E(1), also inhibited the formyl peptide-induced O(2)(-) production. The inhibitory action on the O(2)(-) production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O(2)(-) production.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Superóxidos/antagonistas & inhibidores , Alprostadil/farmacología , Proteínas de Ciclo Celular/metabolismo , Complemento C5a/farmacología , Fibrinolíticos/farmacología , Células HL-60 , Humanos , Concentración de Iones de Hidrógeno , Factores Inmunológicos/farmacología , Isoquinolinas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Superóxidos/metabolismoRESUMEN
GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification.
Asunto(s)
Protones , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Línea Celular , Línea Celular Tumoral , AMP Cíclico/biosíntesis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Lisofosfatidilcolinas/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Elemento de Respuesta al Suero/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.
Asunto(s)
Imidazoles/farmacología , Protones , Piridinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Imidazoles/química , Unión Proteica , Piridinas/química , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Oxidatively damaged proteins and lipid peroxidation products have been shown to accumulate in the brain of neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis, and oxidized lipoprotein is considered to be toxic and neurodegenerative. However, the role of lipoprotein and its oxidized form in neurite remodeling has not been well understood. In the present study, we have aimed to clarify whether and, if so, how high-density lipoprotein (HDL) and oxidized HDL (oxHDL) affect neuritogenesis. In the presence of nerve growth factor, exposure of PC12 cells to either HDL or oxHDL induces a rapid neurite retraction, which is followed by re-outgrowth of neurites in either case; however, oxHDL-treated cells exhibit much longer outgrowths than do basal and HDL-treated cells. Thus, processes in the morphological changes of neuronal cells after lipoprotein treatment are composed of two phases: the reversible retraction phase and the extension phase. Characterization of the active fractions of lipids and experiments with desensitization and knockdown of receptors have indicated that the reversible retraction phase involves mainly sphingosine 1-phosphate for HDL and lysophosphatidic acid for oxHDL. The change in the components responsible for the retraction response is comparable with the change in sphingosine 1-phosphate and lysophosphatidic acid contents by the oxidation of HDL. In the extension phase, lysophosphatidylcholine, which is increased by the oxidation of HDL, may play a stimulatory role in neurite outgrowth. We conclude that lipoprotein and its oxidized form differentially regulate neuritogenesis through lipoprotein-associated lysolipid molecules.
Asunto(s)
Lipoproteínas HDL/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuritas/metabolismo , Animales , Células Cultivadas , Humanos , Lisofosfolípidos/metabolismo , Oxidación-Reducción , Células PC12 , RatasRESUMEN
Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca(2+) pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca(2+) pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of Gq/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/Gq/11 proteins/phospholipase C/Ca(2+)/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.
Asunto(s)
Señalización del Calcio/fisiología , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , GMP Cíclico , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Neuronas/citología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/genética , Péptidos Cíclicos/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Fosfolipasas de Tipo C/genéticaRESUMEN
Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca(2+) response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.