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1.
Am J Respir Cell Mol Biol ; 66(1): 23-37, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34236953

RESUMEN

The U.S. Food and Drug Administration-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential antifibrotic actions. However, neither its in vivo efficacy in lung fibrosis nor its dependence on proteasome inhibition has been conclusively defined. In this study, we assessed the therapeutic efficacy of BTZ in a mouse model of pulmonary fibrosis, developed an in vitro protocol to define its actions on diverse fibroblast activation parameters, determined its reliance on proteasome inhibition for these actions in vivo and in vitro, and explored alternative mechanisms of action. The therapeutic administration of BTZ diminished the severity of pulmonary fibrosis without reducing proteasome activity in the lung. In experiments designed to mimic this lack of proteasome inhibition in vitro, BTZ reduced fibroblast proliferation, differentiation into myofibroblasts, and collagen synthesis. It promoted dedifferentiation of myofibroblasts and overcame their characteristic resistance to apoptosis. Mechanistically, BTZ inhibited kinases important for fibroblast activation while inducing the expression of DUSP1 (dual-specificity protein phosphatase 1), and knockdown of DUSP1 abolished its antifibrotic actions in fibroblasts. Collectively, these findings suggest that BTZ exhibits a multidimensional profile of robust inhibitory actions on lung fibroblasts as well as antifibrotic actions in vivo. Unexpectedly, these actions appear to be independent of proteasome inhibition, instead attributable to the induction of DUSP1.


Asunto(s)
Bortezomib/uso terapéutico , Fibroblastos/patología , Inhibidores de Proteasoma/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , Adulto , Apoptosis/efectos de los fármacos , Bleomicina , Bortezomib/farmacología , Desdiferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Fosfatasa 1 de Especificidad Dual/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Miofibroblastos/efectos de los fármacos , Miofibroblastos/patología , FN-kappa B/metabolismo , Prostaglandinas/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Receptor fas/metabolismo
2.
J Biol Chem ; 292(51): 20897-20910, 2017 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-29101235

RESUMEN

Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.


Asunto(s)
Células Epiteliales Alveolares/fisiología , Comunicación Celular/fisiología , Micropartículas Derivadas de Células/fisiología , Actinas/metabolismo , Lesión Pulmonar Aguda/fisiopatología , Animales , Línea Celular , Dinaminas/metabolismo , Endocitosis , Femenino , Ligandos , Macrófagos Alveolares/fisiología , Modelos Biológicos , Oxidación-Reducción , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Humo/efectos adversos , Nicotiana/toxicidad
3.
Am J Physiol Lung Cell Mol Physiol ; 313(4): L722-L731, 2017 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-28729346

RESUMEN

Prostaglandin E2 (PGE2), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factor ß-1 (TGFß-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. siRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE2 inhibition of either process. However, knockdown of PKARIß selectively attenuated PGE2 inhibition of collagen I protein expression, whereas knockdown of PKARIIß selectively attenuated PGE2 inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIß or PKARIIß exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIß agonist (but not a PKARIIß agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIß agonist (but not a PKARIß agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) as well as the relative mRNA and protein expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflects the actions of distinct PKAR subunits.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/farmacología , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/farmacología , Dinoprostona/antagonistas & inhibidores , Miofibroblastos/citología , Biosíntesis de Proteínas/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , Miofibroblastos/metabolismo , Oxitócicos/farmacología
4.
Am J Respir Cell Mol Biol ; 54(1): 114-27, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26098591

RESUMEN

Myofibroblasts, the major effector cells in pathologic fibrosis, derive from the differentiation of fibroblasts driven by mediators such as transforming growth factor-ß1 (TGF-ß1) and biomechanical signals. Although the myofibroblast has traditionally been considered a terminally differentiated cell, the lipid mediator prostaglandin E2 (PGE2) has been shown to not only prevent but also reverse myofibroblast differentiation, as characterized by the ability of PGE2 to diminish expression of collagen I and α-smooth muscle actin in established myofibroblasts. Here, we use microarrays to examine the extent of transcriptomic changes that occur during TGF-ß1-induced differentiation and PGE2-induced dedifferentiation of myofibroblasts. Normal primary human adult lung fibroblasts were cultured for 24 hours with or without TGF-ß1 and treated for 48 hours with PGE2. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. TGF-ß1 up-regulated 588 genes and down-regulated 689 genes compared with control cells. PGE2 reversed the expression of 363 (62%) of the TGF-ß1-up-regulated genes and 345 (50%) of the TGF-ß1-down-regulated genes. Genes up-regulated by TGF-ß1 and reversed by PGE2 were enriched in annotations for Cell Adhesion, Contractile Fiber, and Actin Binding, whereas genes down-regulated by TGF-ß1 but subsequently reversed by PGE2 were enriched in annotations for Glycoprotein, Polysaccharide Binding, and Regulation of Cell Migration. Surprisingly, the genes whose expression was affected by PGE2 differed between TGF-ß1-induced myofibroblasts and undifferentiated fibroblasts. These data demonstrate the capacity of PGE2 to effect marked global alterations in the transcriptomic program of differentiated myofibroblasts and emphasize the considerable plasticity of these cells.


Asunto(s)
Transdiferenciación Celular/efectos de los fármacos , Dinoprostona/farmacología , Fibroblastos/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Línea Celular , Transdiferenciación Celular/genética , Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genotipo , Humanos , Miofibroblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Mapas de Interacción de Proteínas , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta1/farmacología
5.
Am J Respir Crit Care Med ; 185(1): 77-84, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21940790

RESUMEN

RATIONALE: Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood. OBJECTIVES: This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs. METHODS: Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists. MEASUREMENT AND MAIN RESULTS: PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1ß was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1ß. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio. CONCLUSIONS: These data identify the PGE2 axis as an important autocrine-paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.


Asunto(s)
Bronquiolitis Obliterante/metabolismo , Bronquiolitis Obliterante/patología , Dinoprostona/metabolismo , Trasplante de Pulmón , Pulmón/metabolismo , Pulmón/patología , Actinas/metabolismo , Adenilil Ciclasas/metabolismo , Western Blotting , Líquido del Lavado Bronquioalveolar , Diferenciación Celular , Células Cultivadas , Colágeno/metabolismo , Ciclooxigenasa 2/metabolismo , Fibrosis , Humanos , Interleucina-1beta/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Reacción en Cadena en Tiempo Real de la Polimerasa
6.
Am J Physiol Lung Cell Mol Physiol ; 301(5): L636-44, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21873451

RESUMEN

Asthma is a chronic lung disease characterized by local inflammation that can result in structural alterations termed airway remodeling. One component of airway remodeling involves fibroblast accumulation and activation, resulting in deposition of collagen I around small bronchi. Prostaglandin E(2) (PGE(2)) is the main eicosanoid lipid mediator produced by lung fibroblasts, and it exerts diverse anti-fibrotic actions. Dysregulation of the PGE(2) synthesis/response axis has been identified in human pulmonary fibrotic diseases and implicated in the pathogenesis of animal models of lung parenchymal fibrosis. Here we investigated the relationship between the fibroblast PGE(2) axis and airway fibrosis in an animal model of chronic allergic asthma. Airway fibrosis increased progressively as the number of airway challenges with antigen increased from 3 to 7 to 12. Compared with cells from control lungs, fibroblasts grown from the lungs of asthmatic animals, regardless of challenge number, exhibited no defect in the ability of PGE(2) or its analogs to inhibit cellular proliferation and collagen I expression. This correlated with intact expression of the EP(2) receptor, which is pivotal for PGE(2) responsiveness. However, cytokine-induced upregulation of PGE(2) biosynthesis as well as expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 declined with increasing numbers of antigen challenges. In addition, treatment with the COX-2-selective inhibitor nimesulide potentiated the degree of airway fibrosis following repeated allergen challenge. Because endogenous COX-2-derived PGE(2) acts as a brake on airway fibrosis, the inability of fibroblasts to upregulate PGE(2) generation in the inflammatory milieu presented by repeated allergen exposure could contribute to the airway remodeling and fibrosis observed in chronic asthma.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma , Dinoprostona/biosíntesis , Fibroblastos/metabolismo , Inflamación/fisiopatología , Fibrosis Pulmonar/fisiopatología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Alérgenos/administración & dosificación , Alérgenos/efectos adversos , Animales , Asma/complicaciones , Asma/metabolismo , Asma/patología , Asma/fisiopatología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Colágeno/metabolismo , Ciclooxigenasa 2/biosíntesis , Inhibidores de la Ciclooxigenasa/efectos adversos , Inhibidores de la Ciclooxigenasa/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Histocitoquímica , Humanos , Inflamación/complicaciones , Inflamación/metabolismo , Inflamación/patología , Oxidorreductasas Intramoleculares/biosíntesis , Ratones , Prostaglandina-E Sintasas , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfonamidas/efectos adversos , Sulfonamidas/farmacología
7.
Am J Pathol ; 178(6): 2461-9, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21641374

RESUMEN

Fibrotic obliteration of the small airways leading to progressive airflow obstruction, termed bronchiolitis obliterans syndrome (BOS), is the major cause of poor outcomes after lung transplantation. We recently demonstrated that a donor-derived population of multipotent mesenchymal stem cells (MSCs) can be isolated from the bronchoalveolar lavage (BAL) fluid of human lung transplant recipients. Herein, we study the organ specificity of these cells and investigate the role of local mesenchymal progenitors in fibrogenesis after lung transplantation. We demonstrate that human lung allograft-derived MSCs uniquely express embryonic lung mesenchyme-associated transcription factors with a 35,000-fold higher expression of forkhead/winged helix transcription factor forkhead box (FOXF1) noted in lung compared with bone marrow MSCs. Fibrotic differentiation of MSCs isolated from normal lung allografts was noted in the presence of profibrotic mediators associated with BOS, including transforming growth factor-ß and IL-13. MSCs isolated from patients with BOS demonstrated increased expression of α-SMA and collagen I when compared with non-BOS controls, consistent with a stable in vivo fibrotic phenotype. FOXF1 mRNA expression in the BAL cell pellet correlated with the number of MSCs in the BAL fluid, and myofibroblasts present in the fibrotic lesions expressed FOXF1 by in situ hybridization. These data suggest a key role for local tissue-specific, organ-resident, mesenchymal precursors in the fibrogenic processes in human adult lungs.


Asunto(s)
Trasplante de Pulmón , Pulmón/patología , Células Madre Mesenquimatosas/patología , Actinas/metabolismo , Biomarcadores/metabolismo , Biopsia , Células de la Médula Ósea/patología , Bronquiolitis Obliterante/patología , Líquido del Lavado Bronquioalveolar , Recuento de Células , Diferenciación Celular , Separación Celular , Colágeno/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Pulmón/embriología , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Miofibroblastos/patología , Especificidad de Órganos , Fenotipo , Receptores de Interleucina-13/metabolismo , Trasplante Homólogo
8.
J Clin Invest ; 120(6): 1950-60, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20501949

RESUMEN

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor-1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1-/- mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1-/- mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.


Asunto(s)
Ciclooxigenasa 2/biosíntesis , Dinoprostona/biosíntesis , Inhibidor 1 de Activador Plasminogénico/metabolismo , Plasminógeno/metabolismo , Fibrosis Pulmonar/metabolismo , Adulto , Animales , Bleomicina/efectos adversos , Bleomicina/metabolismo , Bleomicina/farmacología , Colágeno/efectos adversos , Colágeno/metabolismo , Colágeno/farmacología , Dinoprostona/metabolismo , Dinoprostona/farmacología , Matriz Extracelular/metabolismo , Fibrinolisina , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasminógeno/efectos adversos , Plasminógeno/farmacología , Inhibidor 1 de Activador Plasminogénico/efectos adversos , Inhibidor 1 de Activador Plasminogénico/farmacología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Receptor PAR-1/metabolismo
9.
FASEB J ; 23(12): 4317-26, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19671668

RESUMEN

Although the lipid mediator prostaglandin E(2) (PGE(2)) exerts antifibrotic effects by inhibiting multiple fibroblast functions, its ability to regulate fibroblast survival is unknown. Here, we examined the effects of this prostanoid on apoptosis and apoptosis pathways in normal and fibrotic lung fibroblasts. As compared to medium alone, 24 h of treatment with PGE(2) increased apoptosis of normal lung fibroblasts in a dose-dependent manner (EC(50) approximately 50 nM), as measured by annexin V staining, caspase 3 activity, cleavage of poly-ADP-ribose polymerase, and single-stranded DNA levels. PGE(2) also potentiated apoptosis elicited by Fas ligand plus cycloheximide. These proapoptotic actions were dependent on signaling through the EP2/EP4 receptors and by downstream activation of both caspases 8 and 9. Silencing and gene deletion of PTEN demonstrated that the effects of PGE(2) involved decreased activity of the prosurvival molecule Akt. PGE(2) also down-regulated expression of survivin, an inhibitor of apoptosis, and increased expression of Fas. Fibroblasts from patients with pulmonary fibrosis exhibited resistance to the apoptotic effects of PGE(2). These findings show for the first time that, in contrast to its effects on many other cell types, PGE(2) promotes apoptosis in lung fibroblasts through diverse pathways. They provide another dimension by which PGE(2) may inhibit, and perhaps even reverse, fibrogenesis in patients with interstitial lung disease.


Asunto(s)
Apoptosis/efectos de los fármacos , Dinoprostona/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Línea Celular , Regulación de la Expresión Génica , Humanos , Pulmón/citología , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal
10.
J Immunol ; 181(6): 4389-96, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18768898

RESUMEN

Development of allograft rejection continues to be the major determinant of morbidity and mortality postlung transplantation. We have recently demonstrated that a population of donor-derived mesenchymal stem cells is present in human lung allografts and can be isolated and expanded ex vivo. In this study, we investigated the impact of lung resident mesenchymal stem cells (LR-MSCs), derived from allografts of human lung transplant recipients, on T cell activation in vitro. Similar to bone marrow-derived MSCs, LR-MSCs did not express MHC II or the costimulatory molecules CD80 or CD86. In vitro, LR-MSCs profoundly suppressed the proliferative capacity of T cells in response to a mitogenic or an allogeneic stimulus. The immunosuppressive function of LR-MSCs was also noted in the absence of direct cell contact, indicating that LR-MSCs mediated their effect predominantly via a soluble mediator. LR-MSCs isolated from lung transplant recipients demonstrated PGE(2) secretion at baseline (385 +/- 375 pg/ml), which increased in response to IL-1beta (1149 +/- 1081 pg/ml). The addition of PG synthesis inhibitors (indomethacin and NS-398) substantially abrogated LR-MSC-mediated immunosuppression, indicating that PGE(2) may be one of the major soluble mediators impacting T cell activity. This is the first report to demonstrate that human tissue-derived MSCs isolated from an allogeneic environment have the potential to mediate immunological responses in vitro.


Asunto(s)
Proliferación Celular , Dinoprostona/fisiología , Inhibidores de Crecimiento/fisiología , Tolerancia Inmunológica , Trasplante de Pulmón/inmunología , Pulmón/inmunología , Células Madre Mesenquimatosas/inmunología , Subgrupos de Linfocitos T/citología , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Dinoprostona/biosíntesis , Inhibidores de Crecimiento/biosíntesis , Humanos , Pulmón/citología , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Subgrupos de Linfocitos T/inmunología , Trasplante Homólogo
11.
Am J Respir Cell Mol Biol ; 39(4): 482-9, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18421013

RESUMEN

Via their capacities for proliferation and synthesis of matrix proteins such as collagen, fibroblasts are key effectors in the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis. Prostaglandin E(2) (PGE(2)) potently inhibits these functions in lung fibroblasts through receptor ligation and production of the second messenger cAMP, but the downstream pathways mediating such actions have not been fully characterized. We sought to investigate the roles of the cAMP effectors protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1) in modulating these two functions in primary human fetal lung IMR-90 fibroblasts. The specific roles of these two effector pathways were examined by treating cells with PKA-specific (6-bnz-cAMP) and Epac-specific (8-pCPT-2'-O-Me-cAMP) agonists, inhibiting PKA with the inhibitor KT 5720, overexpressing the PKA catalytic subunit, and silencing Epac-1 using short hairpin RNA. PGE(2) inhibition of collagen I expression was mediated exclusively by activation of PKA, while inhibition of fibroblast proliferation was mediated exclusively by activation of Epac-1. PGE(2) and Epac-1 inhibited cell proliferation through activation of the small GTPase Rap1, since decreasing Rap1 activity by transfection with Rap1GAP or the dominant-negative Rap1N17 prevented, and transfection with the constitutively active Rap1V12 mimicked, the anti-proliferative effects of PGE(2). On the other hand, PKA inhibition of collagen was dependent on inhibition of protein kinase C-delta. The selective use of PKA and Epac-1 pathways to inhibit distinct aspects of fibroblast activation illustrate the pleiotropic ability of PGE(2) to inhibit diverse fibroblast functions.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Dinoprostona/farmacología , Fibroblastos/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Pulmón/citología , Carbazoles/farmacología , Dominio Catalítico , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Dinoprostona/fisiología , Activación Enzimática , Fibroblastos/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/agonistas , Humanos , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/metabolismo , Pirroles/farmacología , Proteínas de Unión al GTP rap1/metabolismo
12.
Am J Respir Crit Care Med ; 177(1): 66-74, 2008 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-17916807

RESUMEN

RATIONALE: Prostaglandin (PG) E2, a cyclooxygenase-derived lipid mediator, is a potent down-regulator of fibroblast activation in normal lung fibroblasts. Although fibroblasts from patients with idiopathic pulmonary fibrosis are known to exhibit a defect in PGE2 synthesis, there is little information about their responsiveness to this lipid mediator. OBJECTIVES: To compare responses to PGE2 in normal, usual interstitial pneumonia (UIP), and other diffuse parenchymal lung disease (DPLD) fibroblasts. METHODS: Fibroblasts were grown in vitro from well characterized control (n = 7), UIP (n = 17), or other DPLD (n = 13) lung tissue. The effects of PGE2 on fibroblast proliferation and collagen expression were determined. MEASUREMENTS AND MAIN RESULTS: Only 3 of 12 UIP fibroblast lines exhibited PGE2-mediated inhibition of both collagen synthesis and cell proliferation, as opposed to 6 of 6 nonfibrotic control cell lines. The degree of PGE2 resistance in DPLD fibroblasts was quite variable, with UIP cells exhibiting the greatest degree of resistance to PGE2, whereas other DPLD fibroblasts manifested a degree of resistance intermediate to control and UIP. The resistance to suppression of collagen expression correlated with worse lung function. Molecular mechanisms for resistance included altered E prostanoid receptor profiles and diminished expression of the downstream kinase, protein kinase A. CONCLUSIONS: The recognition that UIP fibroblasts manifest variable refractoriness to PGE2 suppression sheds new light on the activation phenotype of these cells and on the pathogenesis of fibrotic lung disease.


Asunto(s)
Dinoprostona/farmacología , Fibroblastos/efectos de los fármacos , Enfermedades Pulmonares Intersticiales/patología , Adulto , Anciano , Anciano de 80 o más Años , División Celular/efectos de los fármacos , Línea Celular , Colágeno Tipo I/metabolismo , AMP Cíclico/metabolismo , Resistencia a Medicamentos , Femenino , Fibroblastos/patología , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Fibrosis Pulmonar/patología
13.
Am J Physiol Lung Cell Mol Physiol ; 292(2): L405-13, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17028262

RESUMEN

Uncontrolled fibroblast activation is one of the hallmarks of fibrotic lung disease. Prostaglandin E(2) (PGE(2)) has been shown to inhibit fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung. Understanding the mechanisms for these effects may provide insight into the pathogenesis of fibrotic lung disease. Previous work has focused on commercially available fibroblast cell lines derived from tissue whose precise origin and histopathology are often unknown. Here, we sought to define the mechanism of PGE(2) inhibition in patient-derived fibroblasts from peripheral lung verified to be histologically normal. Fibroblasts were grown from explants of resected lung, and proliferation and collagen I expression was determined following treatment with PGE(2) or modulators of its receptors and downstream signaling components. PGE(2) inhibited fibroblast proliferation by 33% and collagen I expression by 62%. PGE(2) resulted in a 15-fold increase in intracellular cAMP; other cAMP-elevating agents inhibited collagen I in a manner similar to PGE(2). These effects were reproduced by butaprost, a PGE(2) analog selective for the cAMP-coupled E prostanoid (EP) 2 receptor, but not by selective EP3 or EP4 agonists. Fibroblasts expressed both major cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1), but only a selective PKA agonist was able to appreciably inhibit collagen I expression. Treatment with okadaic acid, a phosphatase inhibitor, potentiated the effects of PGE(2). Our data indicate that PGE(2) inhibits fibroblast activation in primary lung fibroblasts via binding of EP2 receptor and production of cAMP; inhibition of collagen I proceeds via activation of PKA.


Asunto(s)
Colágeno Tipo I/metabolismo , AMP Cíclico/metabolismo , Fibroblastos/citología , Pulmón/citología , Prostaglandinas E/farmacología , Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/biosíntesis , AMP Cíclico/análogos & derivados , AMP Cíclico/biosíntesis , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/agonistas , Humanos , Pulmón/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E
14.
J Leukoc Biol ; 79(6): 1234-41, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16574771

RESUMEN

Macrophage (MØ) phagocytosis via the Fc receptor for immunoglobulin G (Fc gammaR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that Fc gammaR-mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although Fc gammaR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc gammaR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.


Asunto(s)
Células Dendríticas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucotrieno B4/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgG/fisiología , Animales , Presentación de Antígeno , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/fisiología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas/efectos de los fármacos , Células Dendríticas/enzimología , Células Dendríticas/fisiología , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Activación Enzimática/efectos de los fármacos , Eritrocitos , Hidroxiurea/análogos & derivados , Hidroxiurea/farmacología , Inmunoglobulina G/inmunología , Indoles/farmacología , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/biosíntesis , Macrófagos/enzimología , Macrófagos/fisiología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/fisiología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Proteínas Opsoninas/inmunología , Fagocitosis/fisiología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Ratas , Ratas Wistar , Receptores de Leucotrieno B4/antagonistas & inhibidores , Ovinos , Quinasa Syk
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