RESUMO
Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD.
Assuntos
Autofagossomos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/genética , alfa-Sinucleína/química , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/patologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Modelos Biológicos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Canais de Potássio/deficiência , Cultura Primária de Células , Agregados Proteicos/efeitos dos fármacos , Ratos , alfa-Sinucleína/farmacologiaRESUMO
BACKGROUND & AIMS: The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor. METHODS: Acute pancreatitis was induced in wild-type and Gpbar1(-/-) mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild-type and Gpbar1(-/-) mice were exposed to either submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM). RESULTS: Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS-induced, but not caerulein-induced, pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS, but not when they are induced by exposure to caerulein. CONCLUSIONS: Gpbar1 may play a critical role in the evolution of bile-acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra-acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Doença Aguda , Amilases/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Ceruletídeo/efeitos adversos , Modelos Animais de Doenças , Precursores Enzimáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Receptores Acoplados a Proteínas G/genética , Índice de Gravidade de Doença , Ácido Taurolitocólico/efeitos adversos , Ácido Taurolitocólico/análogos & derivadosRESUMO
Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.
Assuntos
Plaquetas/citologia , Diferenciação Celular/fisiologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Southern Blotting , Western Blotting , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Receptores de Lisoesfingolipídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombopoetina/sangueRESUMO
Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Animais , Linhagem Celular , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50-100 microm) to in situ GBSM rapidly reduced spontaneous Ca(2+) flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 microm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5-1.0 microm) and the K(ATP) channel blocker, glibenclamide (10 microm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1(/) mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP-PKA pathway, and ultimately the opening of K(ATP) channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease.
Assuntos
Ácidos e Sais Biliares/fisiologia , Vesícula Biliar/fisiologia , Canais KATP/metabolismo , Músculo Liso/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Feminino , Vesícula Biliar/inervação , Vesícula Biliar/metabolismo , Cobaias , Interações Hidrofóbicas e Hidrofílicas , Canais KATP/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/inervação , Músculo Liso/metabolismo , Inibição Neural/fisiologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Neuromedin U (Nmu) is a neuropeptide expressed primarily in the gastrointestinal tract and central nervous system. Previous reports have identified two G protein-coupled receptors (designated Nmur1 and Nmur2) that bind Nmu. Recent reports suggest that Nmu mediates immune responses involving mast cells, and Nmur1 has been proposed to mediate these responses. In this study, we generated mice with an Nmur1 deletion and then profiled the responses of these mice in a cutaneous inflammation model utilizing complete Freund's adjuvant (CFA). We report here that mice lacking Nmur1 had normal inflammation responses with moderate changes in serum cytokines compared to Nmur1(+/+) littermates. Although differences in IL-6 were observed in mice lacking Nmu peptide, these mice exhibited a normal response to CFA. Our data argues against a major role for Nmur1 in mediating the reported inflammatory functions of NmU.
Assuntos
Dermatite/imunologia , Receptores de Neurotransmissores/fisiologia , Animais , Citocinas/sangue , Dermatite/genética , Adjuvante de Freund/imunologia , Adjuvante de Freund/farmacologia , Deleção de Genes , Camundongos , Camundongos Knockout , Receptores de Neurotransmissores/genéticaRESUMO
The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7alpha-hydroxylase pathway.
Assuntos
Colesterol/análise , Cálculos Biliares/genética , Cálculos Biliares/metabolismo , Deleção de Genes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Ácidos e Sais Biliares/biossíntese , Colesterol 7-alfa-Hidroxilase/metabolismo , Gorduras na Dieta/metabolismo , Vesícula Biliar/patologia , Cálculos Biliares/química , Regulação da Expressão Gênica , Fígado/patologia , Camundongos , Camundongos Knockout , RNA MensageiroRESUMO
Proxyfan is a histamine H3 receptor protean agonist that can produce a spectrum of pharmacological effects including agonist, inverse agonist, and antagonist. We have discovered that proxyfan (10 mg/kg orally) significantly improved glucose excursion after an ip glucose tolerance test in either lean or high-fat/cholesterol diet-induced obese mice. It also reduced plasma glucose levels comparable to that of metformin (300 mg/kg orally) in a nongenetic type 2 diabetes mouse model. The dose-dependent decrease in glucose excursion correlated with inhibition of ex vivo H3 receptor binding in the cerebral cortex. In addition, glucose levels were significantly reduced compared with vehicle-treated mice after intracerebroventricular administration of proxyfan, suggesting the involvement of central H3 receptors. Proxyfan-induced reduction of glucose excursion was not observed in the H3 receptor knockout mice, suggesting that proxyfan mediates this effect through H3 receptors. Proxyfan reduced glucose excursion by significantly increasing plasma insulin levels in a glucose-independent manner. However, no difference in insulin sensitivity was observed in proxyfan-treated mice. The H1 receptor antagonist chlorpheniramine and the H2 receptor antagonist zolantidine had modest effects on glucose excursion, and neither inhibited the glucose excursion reduced by proxyfan. The H3 receptor antagonist/inverse agonist, thioperamide, had weaker effects on glucose excursion compared with proxyfan, whereas the H3 receptor agonist imetit did not affect glucose excursion. In conclusion, these findings demonstrate, for the first time, that manipulation of central histamine H3 receptor by proxyfan can significantly improve glucose excursion by increasing plasma insulin levels via a glucose-independent mechanism.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Agonistas dos Receptores Histamínicos/farmacologia , Hipoglicemiantes/farmacologia , Imidazóis/farmacologia , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Piperidinas/farmacologia , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismoRESUMO
G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1(-/-) mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1(-/-) mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1(-/-) mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1(-/-) mice on HFD displayed increased hepatic steatosis when compared with Gpbar1(+)(/)(+) males and Gpbar1(-/-) females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.
Assuntos
Gorduras na Dieta/efeitos adversos , Deleção de Genes , Obesidade/etiologia , Obesidade/metabolismo , Receptores Acoplados a Proteínas G/genética , Caracteres Sexuais , Animais , Colesterol/sangue , Modelos Animais de Doenças , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Fígado Gorduroso/epidemiologia , Feminino , Incidência , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/fisiopatologia , Receptores Acoplados a Proteínas G/fisiologia , Fatores de Risco , Triglicerídeos/sangueRESUMO
G protein-coupled receptor 119 (GPR119) is expressed in pancreatic islets and intestine, and is involved in insulin and incretin hormone release. GPR119-knockout (Gpr119(-/-)) mice were reported to have normal islet morphology and normal size, body weight (BW), and fed/fasted glucose levels. However, the physiological function of GPR119 and its role in maintaining glucose homeostasis under metabolic stress remain unknown. Here, we report the phenotypes of an independently generated line of Gpr119(-/-) mice under basal and high-fat diet (HFD)-induced obesity. Under low-fat diet feeding, Gpr119(-/-) mice show normal plasma glucose and lipids, but have lower BWs and lower post-prandial levels of active glucagon-like peptide 1 (GLP-1). Nutrient-stimulated GLP-1 release is attenuated in Gpr119(-/-) mice, suggesting that GPR119 plays a role in physiological regulation of GLP-1 secretion. Under HFD-feeding, both Gpr119(+)(/)(+) and Gpr119(-/-) mice gain weight similarly, develop hyperinsulinemia and hyperleptinemia, but not hyperglycemia or dyslipidemia. Glucose and insulin tolerance tests did not reveal a genotypic difference. These data show that GPR119 is not essential for the maintenance of glucose homeostasis. Moreover, we found that oleoylethanolamide (OEA), reported as a ligand for GPR119, was able to suppress food intake in both Gpr119(+)(/)(+) and Gpr119(-/-) mice, indicating that GPR119 is not required for the hypophagic effect of OEA. Our results demonstrate that GPR119 is important for incretin and insulin secretion, but not for appetite suppression.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Homeostase/genética , Redes e Vias Metabólicas/genética , Receptores Acoplados a Proteínas G/fisiologia , Via Secretória/genética , Animais , Regulação do Apetite/efeitos dos fármacos , Regulação do Apetite/genética , Células Cultivadas , Endocanabinoides , Feminino , Marcação de Genes , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Incretinas/metabolismo , Incretinas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácidos Oleicos/metabolismo , Ácidos Oleicos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Via Secretória/efeitos dos fármacosRESUMO
AIM: To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes. METHODS: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws. RESULTS: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P < 0.01). CONCLUSION: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.
Assuntos
Anti-Inflamatórios/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Ácidos Graxos Voláteis/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Quimiocinas/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Humanos , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , Masculino , Monócitos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: FFAR1/GPR40 is a G-protein-coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid-induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1(-/-)) mice were resistant to high-fat diet-induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid-induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid-induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1(-/-) mice in the C57BL/6 background. RESEARCH DESIGN AND METHODS: We used CL-316,243, a beta3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1(+/+) (wild-type) and Ffar1(-/-) (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity. RESULTS: Insulin secretion was reduced by approximately 50% in Ffar1(-/-) mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1(+/+) and Ffar1(-/-) mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1(-/-) mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1(+/+) and Ffar1(-/-) mice. CONCLUSIONS: FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1(-/-) mice are not protected from high-fat diet-induced insulin resistance or hepatic steatosis.
Assuntos
Gorduras na Dieta/administração & dosagem , Doenças Metabólicas/fisiopatologia , Receptores Acoplados a Proteínas G/fisiologia , Adiposidade , Animais , Peso Corporal/fisiologia , Teste de Tolerância a Glucose , Hiperinsulinismo/fisiopatologia , Insulina/metabolismo , Doenças Metabólicas/etiologia , Doenças Metabólicas/genética , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/genéticaRESUMO
Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.
Assuntos
Quimiocinas CXC/metabolismo , Interleucina-8/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/fisiologia , Sequência de Aminoácidos , Animais , Artrite Experimental/metabolismo , Quimiocina CXCL6 , Clonagem Molecular , Colágeno/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
Mice lacking GPR103A expression display osteopenia. Analysis of mouse quantitative trait loci literature associated with bone mineral density suggested GPR103A ligand P518/Qrfp (chromosome 2qB) as a candidate osteoporosis gene. Promoter and coding regions of mouse P518/Qrfp were sequenced from genomic DNA obtained from the osteoporosis-prone strain SAMP6 and control strains SAMR1, A/J, AKR/J, BALB/c, C3H/HeJ, C57BL/6J, and DBA/2J. Four single-nucleotide polymorphisms (SNPs) were identified in only SAMP6 genomic DNA, g.-1773 T-->C, g.110 A-->G (N37S), g.188 G-->A (R63K), and g.135 T-->C (H45H). The promoter SNP generated a novel neuron-restrictive silencing factor binding site, a repressor that decreases gene expression in nonneuronal tissues. TaqMan analysis demonstrated fivefold lower P518/Qrfp liver expression in SAMP6 versus SAMR1 or C57BL/6J control strains. Tissue distribution of human, mouse, and rat P518/Qrfp and its receptors showed expression in bone and spinal cord. A direct role for P518/Qrfp function in maintaining bone mineral density is suggested.
Assuntos
Doenças Ósseas Metabólicas/genética , Fases de Leitura Aberta/genética , Peptídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Característica Quantitativa Herdável , Receptores Acoplados a Proteínas G/genética , Sequência de Aminoácidos , Animais , Densidade Óssea , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Kaposi's sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively active G protein-coupled receptor known as vGPCR that binds CXC chemokines with high affinity. In this study, we show that conditional transgenic expression of vGPCR by cells of endothelial origin triggers an angiogenic program in vivo, leading to development of an angioproliferative disease that resembles KS. This angiogenic program consists partly in the expression of the angiogenic factors placental growth factor, platelet-derived growth factor B, and inducible NO synthase by the vGPCR-expressing cells. Finally, we show that continued vGPCR expression is essential for progression of the KS-like phenotype and that down-regulation of vGPCR expression results in reduced expression of angiogenic factors and regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS.
Assuntos
Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Receptores de Quimiocinas/fisiologia , Sarcoma de Kaposi/etiologia , Proteínas Virais/fisiologia , Animais , Becaplermina , Modelos Animais de Doenças , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Neovascularização Patológica , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fator de Crescimento Placentário , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas c-sis , Receptores de Quimiocinas/genética , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/patologia , Proteínas Virais/genéticaRESUMO
The KCNN4 potassium-ion channel has been reported to play an important role in regulating antigen-induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM-34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF-alpha and IFN-gamma as well as the message levels of several other pro-inflammatory molecules in the spinal cord. Plasma concentrations of TRAM-34 within a 24-h period were between the in vitro IC(50) and IC(90) values for the KCNN4 channel. The effect of TRAM-34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG-induced EAE in C57BL/6 mice.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Animais , Movimento Celular/imunologia , Movimento Celular/fisiologia , Encefalomielite Autoimune Experimental/prevenção & controle , Inflamação/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Camundongos , RNA Mensageiro/metabolismo , Medula Espinal/imunologia , Medula Espinal/fisiologiaRESUMO
GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.
Assuntos
Genitália/citologia , Genitália/fisiologia , Proteínas/metabolismo , Receptores de Neuropeptídeos/metabolismo , Reprodução/fisiologia , Maturidade Sexual/fisiologia , Animais , Feminino , Genitália/embriologia , Hipogonadismo/patologia , Hipogonadismo/fisiopatologia , Kisspeptinas , Masculino , Camundongos , Camundongos Knockout , Mutação , Especificidade de Órgãos , Fenótipo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/deficiência , Distribuição TecidualRESUMO
The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.
Assuntos
Quimiocinas CC/biossíntese , Coristoma/imunologia , Linfonodos/imunologia , Doenças Linfáticas/imunologia , Camundongos Transgênicos/imunologia , Pâncreas , Pele , Animais , Movimento Celular/imunologia , Quimiocina CCL21 , Quimiocinas CC/genética , Quimiocinas CC/fisiologia , Coristoma/genética , Coristoma/patologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Células de Langerhans/citologia , Células de Langerhans/imunologia , Linfonodos/citologia , Doenças Linfáticas/genética , Doenças Linfáticas/patologia , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Técnicas de Cultura de Órgãos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Pele/citologia , Pele/metabolismo , Transgenes/imunologiaRESUMO
IFN-kappa belongs to a recently identified subclass of type I IFNs. In this study, we report the cloning and preliminary characterization of the murine homologue of IFN-kappa. The gene encodes a 200-aa protein which is 38.5% homologous to human IFN-kappa. Murine IFN-kappa contains four cysteines in analogous positions to those observed in the IFN-alpha and an additional fifth unique cysteine, C174. The murine gene is located on chromosome 4, where other type I murine IFN genes, IFN-alpha and IFN-beta, are clustered. This region is syntenic with human chromosome 9 where the gene encoding IFN-kappa and the type I IFN gene cluster are found. Mouse IFN-kappa is expressed at low levels in peritoneal macrophages and its expression is up-regulated by dsRNA and IFN-gamma. Similar to previously reported transgenic mice carrying type I and type II IFNs, transgenic mice overexpressing murine IFN-kappa in the beta cells of the pancreas develop overt diabetes with hyperglycemia. Histological characterization of pancreatic islets from these transgenic mice showed inflammatory infiltrates with corresponding destruction of beta cells.