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1.
J Biol Chem ; 291(12): 6083-95, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26763235

RESUMO

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.


Assuntos
Fibroblastos/fisiologia , Miosina Tipo II/fisiologia , Fibrose Pulmonar/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Forma Celular , Matriz Extracelular/fisiologia , Feminino , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Fenótipo , Fibrose Pulmonar/patologia
2.
J Immunol ; 196(1): 428-36, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26597012

RESUMO

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.


Assuntos
Lipopolissacarídeos/imunologia , Lesão Pulmonar/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Canais de Cátion TRPV/imunologia , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Matriz Extracelular/metabolismo , Imunoglobulina G/imunologia , Lesão Pulmonar/patologia , Fenômenos Mecânicos , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Fibrose Pulmonar/imunologia , Transdução de Sinais/imunologia
3.
J Clin Invest ; 124(12): 5225-38, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25365224

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-ß; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-ß1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.


Assuntos
Diferenciação Celular , Pulmão/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/metabolismo , Canais de Cátion TRPV/biossíntese , Regulação para Cima , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacologia , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Pulmão/patologia , Camundongos , Camundongos Mutantes , Miofibroblastos/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Canais de Cátion TRPV/genética , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
4.
Respir Res ; 11: 87, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20576117

RESUMO

BACKGROUND: Arginine is an amino acid that serves as a substrate for the enzymes nitric oxide synthase (NOS) and arginase, leading to synthesis of NO and ornithine, respectively. As such, arginine has the potential to influence diverse fundamental processes in the lung. METHODS: We used mice deficient in cationic amino acid transporter (CAT) 2 in models of allergic airway inflammation and pulmonary fibrosis. RESULTS: We report that the arginine transport protein CAT2 was over-expressed in the lung during the induction of allergic airway inflammation. Furthermore, CAT2 mRNA was strongly induced by transgenically over-expressed IL-4, and allergen-induced expression was dependent upon signal-transducer-and-activator-of-transcription (STAT) 6. In situ mRNA hybridization demonstrated marked staining of CAT2, predominantly in scattered mononuclear cells. Analysis of allergic airway inflammation and bleomycin-induced inflammation in CAT2-deficient mice revealed that while inflammation was independent of CAT2 expression, bleomycin-induced fibrosis was dependent upon CAT2. Mechanistic analysis revealed that arginase activity in macrophages was partly dependent on CAT2. CONCLUSION: Taken together, these results identify CAT2 as a regulator of fibrotic responses in the lung.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Asma/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/deficiência , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Arginase/metabolismo , Arginina/metabolismo , Asma/induzido quimicamente , Asma/genética , Asma/imunologia , Bleomicina , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Pulmão/imunologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologia , RNA Mensageiro/metabolismo , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Regulação para Cima
5.
Blood ; 114(13): 2774-82, 2009 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-19641187

RESUMO

The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5'-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase-dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65(-/-) mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner.


Assuntos
Ácidos/farmacologia , AMP Cíclico/fisiologia , Eosinófilos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Asma/complicações , Asma/genética , Asma/metabolismo , Asma/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Feminino , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pneumonia/complicações , Pneumonia/genética , Pneumonia/metabolismo , Pneumonia/patologia , Prótons , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
6.
BMC Immunol ; 10: 33, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19486531

RESUMO

BACKGROUND: Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice. RESULTS: In order to test the hypothesis that arginase has a role in allergic airway inflammation we generated arginase I-deficient bone marrow (BM) chimeric mice. Following transfer of arginase I-deficient BM into irradiated recipient mice, arginase I expression was not required for hematopoietic reconstitution and baseline immunity. Arginase I deficiency in bone marrow-derived cells decreased allergen-induced lung arginase by 85.8 +/- 5.6%. In contrast, arginase II-deficient mice had increased lung arginase activity following allergen challenge to a similar level to wild type mice. BM-derived arginase I was not required for allergen-elicited sensitization, recruitment of inflammatory cells in the lung, and proliferation of cells. Furthermore, allergen-induced airway hyperresponsiveness and collagen deposition were similar in arginase-deficient and wild type mice. Additionally, arginase II-deficient mice respond similarly to their control wild type mice with allergen-induced inflammation, airway hyperresponsiveness, proliferation and collagen deposition. CONCLUSION: Bone marrow cell derived arginase I is the predominant source of allergen-induced lung arginase but is not required for allergen-induced inflammation, airway hyperresponsiveness or collagen deposition.


Assuntos
Alérgenos/imunologia , Arginase , Células da Medula Óssea/enzimologia , Pulmão/metabolismo , Quimera por Radiação , Hipersensibilidade Respiratória/enzimologia , Animais , Arginase/imunologia , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Colágeno/metabolismo , Hiperargininemia , Imunização , Inflamação , Pulmão/imunologia , Pulmão/patologia , Camundongos , Hipersensibilidade Respiratória/patologia
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