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1.
Cell ; 180(2): 278-295.e23, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978345

RESUMO

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismo
3.
Nat Immunol ; 17(9): 1046-56, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27478939

RESUMO

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.


Assuntos
Artrite Juvenil/genética , Doença de Crohn/genética , Infecções/genética , Hanseníase/genética , Macrófagos/imunologia , Proteínas/genética , Choque Séptico/genética , Trifosfato de Adenosina/metabolismo , Animais , Bacteriólise , Células Cultivadas , Metabolismo Energético , Ácido Graxo Sintase Tipo I/metabolismo , Predisposição Genética para Doença , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Oxirredução , Polimorfismo de Nucleotídeo Único , Risco
4.
Cell ; 137(2): 332-43, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19379698

RESUMO

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.


Assuntos
Receptor PAR-1/metabolismo , Trombose/metabolismo , Animais , Plaquetas/metabolismo , Colágeno/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Humanos , Ligantes , Metaloproteinase 1 da Matriz/metabolismo , Estrutura Terciária de Proteína , Receptor PAR-1/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Gastroenterology ; 162(6): 1690-1704, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35031299

RESUMO

BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.


Assuntos
Doença de Crohn , Enterite , Ácidos Graxos Ômega-3 , Animais , Doença de Crohn/tratamento farmacológico , Endorribonucleases , Enterite/induzido quimicamente , Enterite/tratamento farmacológico , Ácidos Graxos Insaturados , Humanos , Inflamação/tratamento farmacológico , Camundongos , Proteínas Serina-Treonina Quinases , Receptor 2 Toll-Like
6.
Gut ; 71(3): 509-520, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33758004

RESUMO

OBJECTIVE: Primary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers. DESIGN: Mice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays. RESULTS: Here, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors. CONCLUSIONS: Activation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages' ability to create a tumour-permissive environment.


Assuntos
Colangite Esclerosante/patologia , Colite Ulcerativa/patologia , Neoplasias do Colo/etiologia , Neovascularização Patológica/etiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Colangite Esclerosante/genética , Colite Ulcerativa/genética , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Macrófagos/fisiologia , Camundongos , Microambiente Tumoral
7.
Nat Immunol ; 8(12): 1303-12, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17965715

RESUMO

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.


Assuntos
Células Endoteliais/fisiologia , Receptor PAR-1/fisiologia , Receptor PAR-2/fisiologia , Sepse/metabolismo , Transdução de Sinais/fisiologia , Animais , Permeabilidade Capilar , Comunicação Celular , Linhagem Celular , Camundongos , Receptor PAR-1/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Sepse/fisiopatologia , Doenças Vasculares/etiologia
8.
Gut ; 66(5): 930-938, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-26858343

RESUMO

OBJECTIVE: Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs. DESIGN: Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes. RESULTS: Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1ß mRNA. CONCLUSIONS: We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.


Assuntos
Fígado Gorduroso Alcoólico/tratamento farmacológico , Lipopeptídeos/farmacologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Caspase 1/metabolismo , Quimiocina CXCL1/genética , Quimiotaxia/efeitos dos fármacos , Fígado Gorduroso Alcoólico/complicações , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Feminino , Células Hep G2 , Hepatite/etiologia , Hepatite/prevenção & controle , Hepatócitos/metabolismo , Humanos , Interleucina-1beta/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipopeptídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Peroxidase/metabolismo , Receptores de Interleucina-8A/sangue , Taxa de Sobrevida , Transcrição Gênica/efeitos dos fármacos , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/genética , Redução de Peso/efeitos dos fármacos
10.
Blood ; 119(7): 1717-25, 2012 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-22186993

RESUMO

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.


Assuntos
Leucemia Linfoide/tratamento farmacológico , Lipopeptídeos/uso terapêutico , Linfoma/tratamento farmacológico , Receptores CXCR4/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patologia , Lipopeptídeos/administração & dosagem , Lipopeptídeos/síntese química , Lipopeptídeos/química , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Moleculares , Terapia de Alvo Molecular , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Med ; 11(6): 661-5, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15880119

RESUMO

We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides-termed pepducins-that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.


Assuntos
Peptídeos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Humanos , Interleucina-8/fisiologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Conformação Proteica , Subunidades Proteicas , Receptores CXCR4/fisiologia , Receptores de Interleucina-8A/química , Receptores de Interleucina-8A/fisiologia , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/fisiologia
12.
Cell Metab ; 34(1): 106-124.e10, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34986329

RESUMO

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.


Assuntos
Autoimunidade , Purinas , Linfócitos T CD8-Positivos , Células Dendríticas , Ativação Linfocitária , Purinas/metabolismo
13.
Thromb J ; 8(1): 2, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-20181055

RESUMO

BACKGROUND: Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown. RESULTS: Fibrinogen and its digestion fragment D induced pro-coagulant activation of monocytes as assessed in a cellular coagulation assay by reductions in clotting times. Pro-coagulant activation was reversed by blocking antibodies against Mac-1 or LFA-1. Pre-exposure of monocytes to the p38 MAPK inhibitor SB 202190 and the MEK1.2 inhibitor U0126 led to significant increasees in coagulation times whereas blocking JNKII with its inhibitor had no such effect. Blocking NFkappaB with MG-132 also inhibited pro-coagulant activation of monocytes by fibrinogen. A selective inhibitor of matrix metalloproteinase-9 increased times to clot formation whereas other matrix metalloproteinase inhibitors did not significantly interfere with fibrinogen-augmented clot formation in this assay. Treatment of monocytes with fibrinogen increased concentrations of matrix metalloproteinase-9 immunoreactivity in their supernatants. CONCLUSIONS: Fibrinogen induces monocyte pro-coagulant activation in an integrin-, nuclear factor kappaB-, p38 MAPK-, and MEK1.2-dependent manner. Activation of monocytes by fibrinogen increases metalloproteinase-9 secretion, metalloproteinase-9 itself enhances monocyte coagulation by an autocrine mechanism. Results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways.

14.
Mol Cancer Ther ; 7(9): 2746-57, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18790755

RESUMO

Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer.


Assuntos
Ascite/enzimologia , Metaloproteases/antagonistas & inibidores , Neovascularização Patológica/enzimologia , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/patologia , Receptor PAR-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Líquidos Corporais/enzimologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Docetaxel , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Oligopeptídeos/administração & dosagem , Oligopeptídeos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Peritônio/enzimologia , Peritônio/patologia , Taxoides/administração & dosagem , Taxoides/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Sci Signal ; 12(562)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30600262

RESUMO

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.


Assuntos
Proliferação de Células , Glicólise , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Carcinogênese , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas G/genética , ATPase Trocadora de Sódio-Potássio/genética , Células THP-1 , Quinases da Família src/genética , Quinases da Família src/metabolismo
16.
Circulation ; 113(9): 1244-54, 2006 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-16505172

RESUMO

BACKGROUND: Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. METHODS AND RESULTS: Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. CONCLUSIONS: We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.


Assuntos
Plaquetas , Fragmentos de Peptídeos/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptores de Trombina/antagonistas & inibidores , Trombose/tratamento farmacológico , Animais , Linhagem Celular , Quimiotaxia , Dimerização , Modelos Animais de Doenças , Quimioterapia Combinada , Cobaias , Hirudinas/farmacologia , Humanos , Fragmentos de Peptídeos/uso terapêutico , Agregação Plaquetária , Ligação Proteica , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Trombina/metabolismo , Trombose/etiologia , Transfecção
18.
FEBS J ; 273(19): 4416-24, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16956369

RESUMO

Inflammation is traditionally viewed as a physiological reaction to tissue injury. Leukocytes contribute to the inflammatory response by the secretion of cytotoxic and pro-inflammatory compounds, by phagocytotic activity and by targeted attack of foreign antigens. Leukocyte accumulation in tissues is important for the initial response to injury. However, the overzealous accumulation of leukocytes in tissues also contributes to a wide variety of diseases, such as atherosclerosis, chronic inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, vasculitis, systemic inflammatory response syndrome, juvenile diabetes and psoriasis. Many therapeutic interventions target immune cells after they have already migrated to the site of inflammation. This review addresses different therapeutic strategies, used to reduce or prevent leukocyte-endothelial cell interactions and communication, in order to limit the progression of inflammatory diseases.


Assuntos
Comunicação Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Inflamação/tratamento farmacológico , Leucócitos/fisiologia , Animais , Humanos , Inflamação/etiologia , Integrinas/antagonistas & inibidores , Integrinas/fisiologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/fisiologia , Selectinas/efeitos dos fármacos , Selectinas/fisiologia
19.
Blood Coagul Fibrinolysis ; 17(7): 521-6, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16988545

RESUMO

The objective was to estimate the effect of antithrombin therapy on mortality in disseminated intravascular coagulation (DIC) of severe sepsis and septic shock. Randomized clinical trials (RCT) on patients with DIC and severe sepsis or septic shock assigned to intravenous antithrombin or placebo were searched. Eligible studies reported death as the outcome measure. Of 35 RCT, 32 trials were excluded because patients were not randomized to antithrombin versus placebo, or no separate data on patients with DIC were given. In three RCT, 364 patients with severe sepsis or septic shock and DIC were randomized. The disease severity, definition of DIC, dose and duration of treatment varied among the trials. In two of the three RCT, data were from subgroup analyses (patients not stratified by DIC). The combined odds ratio for short-term all-cause mortality in those who received antithrombin was 0.649 (95% confidence interval, 0.422-0.998). Data on bleeding complications in patients treated with antithrombin were reported only in one of the RCT and were not considered suitable for systematic safety estimation. In sepsis patients with DIC, administration of antithrombin concentrate may increase overall survival. Current available evidence, however, is not suited to sufficiently inform clinical practice.


Assuntos
Antitrombinas/uso terapêutico , Coagulação Intravascular Disseminada/tratamento farmacológico , Sepse/complicações , Choque Séptico/complicações , Antitrombinas/efeitos adversos , Coagulação Intravascular Disseminada/etiologia , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Sepse/tratamento farmacológico , Choque Séptico/tratamento farmacológico , Resultado do Tratamento
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