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1.
Sci Rep ; 10(1): 6754, 2020 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-32317758

RESUMEN

Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4+ T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4+ T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Pulmón/inmunología , Músculo Liso/inmunología , Proteína Wnt-5a/inmunología , Actinas/genética , Actinas/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Alérgenos/administración & dosificación , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Movimiento Celular , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Regulación de la Expresión Génica , Humanos , Inmunoglobulina E/biosíntesis , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-5/genética , Interleucina-5/inmunología , Interleucinas/genética , Interleucinas/inmunología , Pulmón/efectos de los fármacos , Pulmón/patología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Transgénicos , Músculo Liso/química , Músculo Liso/patología , Ovalbúmina/administración & dosificación , Cultivo Primario de Células , Transgenes , Proteína Wnt-5a/genética , Proteína Wnt-5a/farmacología
2.
Br J Pharmacol ; 173(23): 3327-3341, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27629364

RESUMEN

BACKGROUND AND PURPOSE: Asthma is a heterogeneous chronic inflammatory disease, characterized by the development of structural changes (airway remodelling). ß-catenin, a transcriptional co-activator, is fundamentally involved in airway smooth muscle growth and may be a potential target in the treatment of airway smooth muscle remodelling. EXPERIMENTAL APPROACH: We assessed the ability of small-molecule compounds that selectively target ß-catenin breakdown or its interactions with transcriptional co-activators to inhibit airway smooth muscle remodelling in vitro and in vivo. KEY RESULTS: ICG-001, a small-molecule compound that inhibits the ß-catenin/CREB-binding protein (CBP) interaction, strongly and dose-dependently inhibited serum-induced smooth muscle growth and TGFß1-induced production of extracellular matrix components in vitro. Inhibition of ß-catenin/p300 interactions using IQ-1 or inhibition of tankyrase 1/2 using XAV-939 had considerably less effect. In a mouse model of allergic asthma, ß-catenin expression in the smooth muscle layer was found to be unaltered in control versus ovalbumin-treated animals, a pattern that was found to be similar in smooth muscle within biopsies taken from asthmatic and non-asthmatic donors. However, ß-catenin target gene expression was highly increased in response to ovalbumin; this effect was prevented by topical treatment with ICG-001. Interestingly, ICG-001 dose-dependently reduced airway smooth thickness after repeated ovalbumin challenge, but had no effect on the deposition of collagen around the airways, mucus secretion or eosinophil infiltration. CONCLUSIONS AND IMPLICATIONS: Together, our findings highlight the importance of ß-catenin/CBP signalling in the airways and suggest ICG-001 may be a new therapeutic approach to treat airway smooth muscle remodelling in asthma.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Pirimidinonas/farmacología , Animales , Antiasmáticos/administración & dosificación , Asma/fisiopatología , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Proteína de Unión a CREB/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Músculo Liso/metabolismo , Ovalbúmina/inmunología , Pirimidinonas/administración & dosificación , beta Catenina/genética , beta Catenina/metabolismo
3.
Am J Physiol Lung Cell Mol Physiol ; 311(3): L529-37, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27422998

RESUMEN

Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-ß1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-ß1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-ß1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-ß1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-ß1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-ß1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.


Asunto(s)
Miocitos del Músculo Liso/metabolismo , Transactivadores/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Transporte Activo de Núcleo Celular , Remodelación de las Vías Aéreas (Respiratorias) , Células Cultivadas , Humanos , Contracción Muscular , Músculo Liso/metabolismo , Músculo Liso/patología , Quinasas Asociadas a rho/metabolismo
4.
Am J Physiol Lung Cell Mol Physiol ; 310(11): L1166-76, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27036869

RESUMEN

COPD is a progressive chronic lung disease characterized by pulmonary inflammation. Several recent studies indicate aberrant expression of WNT ligands and Frizzled receptors in the disease. For example, WNT-5A/B ligand expression was recently found to be increased in lung fibroblasts of COPD patients. However, possible effects of WNT-5A and WNT-5B on inflammation have not been investigated yet. In this study, we assessed the regulation of inflammatory cytokine release in response to WNT-5A/B signaling in human lung fibroblasts. Primary human fetal lung fibroblasts (MRC-5), and primary lung fibroblasts from COPD patients and non-COPD controls were treated with recombinant WNT-5A or WNT-5B to assess IL-6 and CXCL8 cytokine secretion and gene expression levels. Following WNT-5B, and to a lesser extent WNT-5A stimulation, fibroblasts showed increased IL-6 and CXCL8 cytokine secretion and mRNA expression. WNT-5B-mediated IL-6 and CXCL8 release was higher in fibroblasts from COPD patients than in non-COPD controls. In MRC-5 fibroblasts, WNT-5B-induced CXCL8 release was mediated primarily via the Frizzled-2 receptor and TAK1 signaling, whereas canonical ß-catenin signaling was not involved. In further support of noncanonical signaling, we showed activation of JNK, p38, and p65 NF-κB by WNT-5B. Furthermore, inhibition of JNK and p38 prevented WNT-5B-induced IL-6 and CXCL8 secretion, whereas IKK inhibition prevented CXCL8 secretion only, indicating distinct pathways for WNT-5B-induced IL-6 and CXCL8 release. WNT-5B induces IL-6 and CXCL8 secretion in pulmonary fibroblasts. In summary, WNT-5B mediates this via Frizzled-2 and TAK1. As WNT-5 signaling is increased in COPD, this WNT-5-induced inflammatory response could represent a therapeutic target.


Asunto(s)
Fibroblastos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Estudios de Casos y Controles , Línea Celular , Fibroblastos/inmunología , Receptores Frizzled/metabolismo , Expresión Génica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Enfermedad Pulmonar Obstructiva Crónica/inmunología
5.
Respir Res ; 17: 13, 2016 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-26846267

RESUMEN

BACKGROUND: The long-acting anticholinergic tiotropium has recently been registered for the treatment of asthma, and its use is associated with a reduction in exacerbation frequency. Anti-inflammatory and anti-remodeling effects of tiotropium have been demonstrated in in vitro and in vivo models. Because tiotropium treatment is used in combination with inhaled corticosteroids, potential additive effects between the two would be clinically relevant. Therefore, the aim of this study was to investigate additive effects between tiotropium and ciclesonide on airway inflammation and remodeling in guinea pig models of asthma. METHODS: Guinea pigs (n = 3-8/group) were sensitized and challenged with ovalbumin in an acute (single challenge) and a chronic model (12 weekly challenges) of allergic asthma. Animals were treated with vehicle, nebulized tiotropium (0.01-0.3 mM) and/or intranasally instilled ciclesonide (0.001-1 mg/kg) before each challenge. Bronchoalveolar lavage fluid and lungs were collected for analysis of airway inflammation and remodeling. RESULTS: Tiotropium and ciclesonide treatment, alone or in combination, did not inhibit airway inflammation in the acute asthma model. In a dose-finding study, low doses of tiotropium and ciclesonide inhibited airway eosinophilia and airway smooth muscle thickening in the chronic asthma model. Threshold doses of 0.01 mM tiotropium (nebulizer concentration) and 0.01 mg/kg ciclesonide were selected to investigate potential additive effects between both drugs. At these doses, tiotropium and ciclesonide did not inhibit airway eosinophilia or airway smooth muscle thickening when administered alone, but significantly inhibited these allergen-induced responses when administered in combination. CONCLUSIONS: Combined treatment with low doses of tiotropium and ciclesonide inhibits airway inflammation and remodeling in a guinea pig model of chronic asthma, suggesting that combined treatment with anticholinergics and corticosteroids may have anti-inflammatory and anti-remodeling activity in allergic airway diseases. Since tiotropium is registered as a therapy for asthma added on to corticosteroid treatment, these beneficial effects of the combination therapy may be clinically relevant.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Asma/inmunología , Asma/prevención & control , Modelos Animales de Enfermedad , Pregnenodionas/administración & dosificación , Bromuro de Tiotropio/administración & dosificación , Administración por Inhalación , Animales , Antialérgicos/administración & dosificación , Asma/inducido químicamente , Broncodilatadores/administración & dosificación , Enfermedad Crónica , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada/métodos , Cobayas , Masculino , Ovalbúmina , Resultado del Tratamiento
6.
Thorax ; 71(4): 312-22, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26797711

RESUMEN

RATIONALE: We have previously shown increased expression of the Frizzled-8 receptor of the Wingless/integrase-1 (WNT) signalling pathway in COPD. Here, we investigated if the Frizzled-8 receptor has a functional role in airway inflammation associated with chronic bronchitis. METHODS: Acute cigarette-smoke-induced airway inflammation was studied in wild-type and Frizzled-8-deficient mice. Genetic association studies and lung expression quantitative trait loci (eQTL) analyses for Frizzled-8 were performed to evaluate polymorphisms in FZD8 and their relationship to tissue expression in chronic bronchitis. Primary human lung fibroblasts and primary human airway epithelial cells were used for in vitro studies. RESULTS: Cigarette-smoke-exposure induced airway inflammation in wild-type mice, which was prevented in Frizzled-8-deficient mice, suggesting a crucial role for Frizzled-8 in airway inflammation. Furthermore, we found a significant genetic association (p=0.009) between single nucleotide polymorphism (SNP) rs663700 in the FZD8 region and chronic mucus hypersecretion, a characteristic of chronic bronchitis, in a large cohort of smoking individuals. We found SNP rs663700 to be a cis-eQTL regulating Frizzled-8 expression in lung tissue. Functional data link mesenchymal Frizzled-8 expression to inflammation as its expression in COPD-derived lung fibroblasts was regulated by pro-inflammatory cytokines in a genotype-dependent manner. Moreover, Frizzled-8 regulates inflammatory cytokine secretion from human lung fibroblasts, which in turn promoted MUC5AC expression by differentiated human airway epithelium. CONCLUSIONS: These findings indicate an important pro-inflammatory role for Frizzled-8 and suggest that its expression is related to chronic bronchitis. Furthermore, our findings indicate an unexpected role for fibroblasts in regulating airway inflammation in COPD.


Asunto(s)
Bronquitis Crónica/genética , Receptores Frizzled/genética , Polimorfismo de Nucleótido Simple/genética , Animales , Bronquitis Crónica/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Marcadores Genéticos/genética , Genotipo , Humanos , Técnicas In Vitro , Inflamación/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucina 5AC/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Transducción de Señal/genética
7.
Front Physiol ; 7: 657, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28101062

RESUMEN

Background: COPD is a progressive lung disease characterized by emphysema and enhanced bronchoconstriction. Current treatments focused on bronchodilation can delay disease progression to some extent, but recovery or normalization of loss of lung function is impossible. Therefore, novel therapeutic targets are needed. The importance of the parenchyma in airway narrowing is increasingly recognized. In COPD, the parenchyma and extracellular matrix are altered, possibly affecting airway mechanics and enhancing bronchoconstriction. Our aim was to set up a comprehensive ex vivo Precision Cut Lung Slice (PCLS) model with a pathophysiology resembling that of COPD and integrate multiple readouts in order to study the relationship between parenchyma, airway functionality, and lung repair processes. Methods: Lungs of C57Bl/6J mice were sliced and treated ex vivo with elastase (2.5 µg/ml) or H2O2 (200 µM) for 16 h. Following treatment, parenchymal structure, airway narrowing, and gene expression levels of alveolar Type I and II cell repair were assessed. Results: Following elastase, but not H2O2 treatment, slices showed a significant increase in mean linear intercept (Lmi), reflective of emphysema. Only elastase-treated slices showed disorganization of elastin and collagen fibers. In addition, elastase treatment lowered both alveolar Type I and II marker expression, whereas H2O2 stimulation lowered alveolar Type I marker expression only. Furthermore, elastase-treated slices showed enhanced methacholine-induced airway narrowing as reflected by increased pEC50 (5.87 at basal vs. 6.50 after elastase treatment) and Emax values (47.96 vs. 67.30%), and impaired chloroquine-induced airway opening. The increase in pEC50 correlated with an increase in mean Lmi. Conclusion: Using this model, we show that structural disruption of elastin fibers leads to impaired alveolar repair, disruption of the parenchymal compartment, and altered airway biomechanics, enhancing airway contraction. This finding may have implications for COPD, as the amount of elastin fiber and parenchymal tissue disruption is associated with disease severity. Therefore, we suggest that PCLS can be used to model certain aspects of COPD pathophysiology and that the parenchymal tissue damage observed in COPD contributes to lung function decline by disrupting airway biomechanics. Targeting the parenchymal compartment may therefore be a promising therapeutic target in the treatment of COPD.

8.
Am J Physiol Lung Cell Mol Physiol ; 308(3): L301-6, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25480330

RESUMEN

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T3) and l-thyroxine (T4) in the absence and presence of the profibrotic transforming growth factor (TGF)-ß1 on human ASM cell phenotype switching. T3 (1-100 nM) and T4 (1-100 nM) did not affect basal ASM proliferation. However, when combined with TGF-ß1 (2 ng/ml), T4 synergistically increased the proliferative response, whereas only a minor effect was observed for T3. In line with a switch from a contractile to a proliferative ASM phenotype, T4 reduced the TGF-ß1-induced contractile protein expression by ∼50%. Cotreatment with T3 reduced TGF-ß1-induced contractile protein expression by ∼25%. The synergistic increase in proliferation was almost fully inhibited by the integrin αvß3 antagonist tetrac (100 nM), whereas no significant effects of the thyroid receptor antagonist 1-850 (3 µM) were observed. Inhibition of MEK1/2, downstream of the integrin αvß3, also inhibited the T4- and TGF-ß1-induced proliferative responses. Collectively, the results indicate that T4, and to a lesser extent T3, promotes a proliferative ASM phenotype in the presence of TGF-ß1, which is predominantly mediated by the membrane-bound T4 receptor αvß3. These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease.


Asunto(s)
Miocitos del Músculo Liso/fisiología , Tiroxina/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Remodelación de las Vías Aéreas (Respiratorias) , Bronquios/patología , Línea Celular , Proliferación Celular , Regulación hacia Abajo , Expresión Génica , Humanos , Integrina alfaVbeta3/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fenotipo , Triyodotironina/fisiología
9.
PLoS One ; 9(4): e94801, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24728340

RESUMEN

WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-ß in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-ß-induced WNT-5A expression. We show that TGF-ß-activated kinase 1 (TAK1) is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-ß induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK) signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-ß-induced WNT-5A expression. Remarkably, we demonstrate a central role of ß-catenin in TGF-ß-induced WNT-5A expression. Regulated by TAK1, ß-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with ß-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-ß-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, ß-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-ß which regulates WNT-5A induction.


Asunto(s)
Regulación de la Expresión Génica , Quinasas Quinasa Quinasa PAM/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/genética , beta Catenina/metabolismo , Línea Celular Transformada , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Proteína Wnt-5a , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
10.
Br J Pharmacol ; 169(3): 590-603, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23297769

RESUMEN

BACKGROUND: Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-ß is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-ß1-induced myofibroblast differentiation is currently largely unknown. PURPOSE: To determine the contribution of GSK-3 signalling in TGF-ß1-induced myofibroblast differentiation. EXPERIMENTAL APPROACH: We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. RESULTS: Stimulation of MRC5 and primary human lung fibroblasts with TGF-ß1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-ß1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-ß1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-ß/smad signalling. CONCLUSION AND IMPLICATION: We demonstrate that GSK-3 signalling regulates TGF-ß1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.


Asunto(s)
Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/agonistas , Fibroblastos/citología , Glucógeno Sintasa Quinasa 3/metabolismo , Pulmón/citología , Miofibroblastos/citología , Factor de Crecimiento Transformador beta1/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética
11.
FASEB J ; 27(4): 1631-43, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23254341

RESUMEN

Transforming growth factor ß (TGF-ß), a key mediator of fibrotic responses, is increased in asthma and drives airway remodeling by inducing expression of extracellular matrix (ECM) proteins. We investigated the molecular mechanisms underlying TGF-ß-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-ß and Wingless/integrase 1 (WNT) signaling in ECM deposition. Airway smooth muscle expresses abundant WNT ligands, with the noncanonical WNT-5A being the most profoundly expressed. Interestingly, WNT-5A shows ∼2-fold higher abundance in airway smooth muscle cells isolated from individuals with asthma than individuals without asthma. WNT-5A is markedly induced in response to TGF-ß (4-16-fold; EC50 0.3 ng/ml) and is required for collagen and fibronectin expression by airway smooth muscle. WNT-5A engages noncanonical WNT signaling pathways, as inhibition of Ca(2+) and c-Jun N-terminal kinase (JNK) signaling attenuated this TGF-ß response, whereas the canonical WNT antagonist Dickkopf 1 (DKK-1) did not. Accordingly, WNT-5A induced JNK phosphorylation and nuclear translocation of nuclear factor of activated T cells c1 (NFATc1). Furthermore, silencing of the WNT-5A receptors Frizzled 8 (FZD8) and RYK attenuated TGF-ß-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-ß-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Calcio/metabolismo , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Proteína Wnt-5a
12.
PLoS One ; 7(2): e31574, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22363678

RESUMEN

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the ß(2)-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1.


Asunto(s)
Antiinflamatorios/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Anciano , Anciano de 80 o más Años , Bronquios/patología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fenoterol/farmacología , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Inhibidor NF-kappaB alfa , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Transducción de Señal/efectos de los fármacos , Fumar , Factor de Transcripción ReIA/metabolismo
13.
Am J Physiol Lung Cell Mol Physiol ; 301(6): L956-65, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21908588

RESUMEN

Chronic inflammatory airway diseases like asthma and chronic obstructive pulmonary disease (COPD) are characterized by airway remodeling with altered extracellular matrix (ECM) deposition. Transforming growth factor-ß(1) (TGF-ß(1)) is upregulated in asthma and COPD and contributes to tissue remodeling in the airways by driving ECM production by structural cells, including airway smooth muscle. In this study, we investigated the activation of ß-catenin signaling and its contribution to ECM production by airway smooth muscle cells in response to TGF-ß(1). Stimulation of airway smooth muscle cells with TGF-ß(1) resulted in a time-dependent increase of total and nonphosphorylated ß-catenin protein expression via induction of ß-catenin mRNA and inhibition of GSK-3. In addition, the TGF-ß(1)-induced ß-catenin activated TCF/LEF-dependent gene transcription, as determined by the ß-catenin sensitive TOP-flash luciferase reporter assay. Furthermore, TGF-ß(1) stimulation increased mRNA expression of collagen Iα1, fibronectin, versican, and PAI-1. Pharmacological inhibition of ß-catenin by PKF115-584 or downregulation of ß-catenin expression by specific small interfering RNA (siRNA) substantially inhibited TGF-ß(1)-induced expression of the ECM genes. Fibronectin protein deposition by airway smooth muscle cells in response to TGF-ß(1) was also inhibited by PKF115-584 and ß-catenin siRNA. Moreover, transfection of airway smooth muscle cells with a nondegradable ß-catenin mutant (S33Y ß-catenin) was sufficient for inducing fibronectin protein expression. Collectively, these findings indicate that ß-catenin signaling is activated in response to TGF-ß(1) in airway smooth muscle cells, which is required and sufficient for the regulation of ECM protein production. Targeting ß-catenin-dependent gene transcription may therefore hold promise as a therapeutic intervention in airway remodeling in both asthma and COPD.


Asunto(s)
Matriz Extracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/farmacología , beta Catenina/metabolismo , Bronquios/patología , Línea Celular , Fibronectinas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Enfermedades Pulmonares Obstructivas/metabolismo , Enfermedades Pulmonares Obstructivas/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Perileno/análogos & derivados , Perileno/farmacología , Interferencia de ARN , Transcripción Genética , Activación Transcripcional , Factor de Crecimiento Transformador beta1/fisiología , beta Catenina/antagonistas & inhibidores , beta Catenina/genética
14.
Br J Pharmacol ; 164(3): 958-69, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21426315

RESUMEN

BACKGROUND AND PURPOSE: Platelet-derived growth factor (PDGF) modulates the airway smooth muscle (ASM) 'contractile' phenotype to a more 'proliferative' phenotype, resulting in increased proliferation and reduced contractility. Such phenotypic modulation may contribute to airway remodelling in asthma. We have previously shown that the cAMP effector molecules, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) inhibited PDGF-induced phenotypic modulation in bovine ASM. Here, we investigated these mechanisms in human ASM strips and cells. EXPERIMENTAL APPROACH: ASM strips were incubated with PDGF in the absence or presence of the activators of Epac (8-pCPT-2'-O-Me-cAMP) or of PKA (6-Bnz-cAMP) for 4 days. Strips were mounted for isometric contraction experiments or analysed for the expression of contractile markers. Cell proliferation was measured and proliferative markers were analysed under similar conditions. KEY RESULTS: Activation of Epac and PKA prevented PDGF-induced ASM strip hypocontractility, and restored the expression of smooth muscle actin, myosin and calponin, which had been markedly diminished by PDGF. Epac and PKA activation inhibited the PDGF-induced ASM cell proliferation and G(1)/S phase transition and the expression and phosphorylation of cell cycle regulators. CONCLUSIONS AND IMPLICATIONS: Epac and PKA maintain a normally contractile ASM phenotype in a mitogenic environment, suggesting that specific activators of Epac and PKA may be beneficial in the treatment of airway remodelling in asthma.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Músculo Liso/metabolismo , Tráquea/metabolismo , Actinas/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular , Humanos , Proteínas de Microfilamentos/metabolismo , Contracción Muscular/fisiología , Músculo Liso/citología , Músculo Liso/enzimología , Miosinas/metabolismo , Fenotipo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tráquea/citología , Tráquea/enzimología , Calponinas
15.
Am J Physiol Lung Cell Mol Physiol ; 300(6): L910-9, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21421749

RESUMEN

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signaling proteins and transcription factors involved in inflammation. Its role in inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), is largely unknown. We investigated the role of GSK-3 in the secretion of chemokines and growth factors by human airway smooth muscle cells after exposure to cigarette smoke extract (CSE) or interleukin-1ß (IL-1ß), important factors involved in the development of COPD. Cultured human airway smooth muscle cells were exposed to increasing concentrations of CSE (1-15%) and IL-1ß (0.01-1.0 ng/ml), which induced the secretion of VEGF-A and IL-8, whereas eotaxin secretion was induced by IL-1ß only. Inhibition of GSK-3 by the selective inhibitor SB216763 or CHIR/CT99021 attenuated the cytokine and growth factor release induced by CSE and/or IL-1ß, without affecting the basal release. Secretion of the cytokines by airway smooth muscle partially depends on NF-κB signaling, and GSK-3 has been implicated in regulating multiple steps in activating the NF-κB signaling pathway. IL-1ß treatment induced degradation of the NF-κB inhibitory protein Iκ-Bα followed by nuclear translocation and DNA binding of p65 NF-κB, which were unaffected by inhibition of GSK-3. However, induction of NF-κB-dependent transcriptional activity by IL-1ß and CSE was largely reduced upon GSK-3 inhibition by SB216763. Collectively, we demonstrate that CSE and IL-1ß activate airway smooth muscle cells to secrete the proinflammatory cytokines IL-8, eotaxin, and VEGF-A. Furthermore, we show that GSK-3 regulates the release of these cytokines induced by CSE and IL-1ß by promoting NF-κB-dependent gene transcription.


Asunto(s)
Bronquios/efectos de los fármacos , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Interleucina-1beta/farmacología , Músculo Liso/efectos de los fármacos , Fumar , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Músculo Liso/citología , Músculo Liso/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos
16.
Br J Pharmacol ; 162(1): 193-209, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20804494

RESUMEN

BACKGROUND AND PURPOSE: Changes in airway smooth muscle (ASM) phenotype may contribute to the pathogenesis of airway disease. Platelet-derived growth factor (PDGF) switches ASM from a contractile to a proliferative, hypo-contractile phenotype, a process requiring activation of extracellular signal-regulated kinase (ERK) and p70(S6) Kinase (p70(S6K) ). The effects of cAMP-elevating agents on these processes is unknown. Here, we investigated the effects of cAMP elevation by prostaglandin E(2) (PGE(2) ) and the activation of the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (Epac) on PDGF-induced phenotype switching in bovine tracheal smooth muscle (BTSM). EXPERIMENTAL APPROACH: Effects of long-term treatment with the PGE(2) analogue 16,16-dimethyl-PGE(2) , the selective Epac activator, 8-pCPT-2'-O-Me-cAMP and the selective PKA activator, 6-Bnz-cAMP were assessed on the induction of a hypo-contractile, proliferative BTSM phenotype and on activation of ERK and p70(S6K) , both induced by PDGF. KEY RESULTS: Treatment with 16,16-dimethyl-PGE(2) inhibited PDGF-induced proliferation of BTSM cells and maintained BTSM strip contractility and contractile protein expression in the presence of PDGF. Activation of both Epac and PKA similarly prevented PDGF-induced phenotype switching and PDGF-induced activation of ERK. Interestingly, only PKA activation resulted in inhibition of PDGF-induced phosphorylation of p70(S6K) . CONCLUSIONS AND IMPLICATIONS: Our data indicate for the first time that both Epac and PKA regulated switching of ASM phenotype via differential inhibition of ERK and p70(S6K) pathways. These findings suggest that cAMP elevation may be beneficial in the treatment of long-term changes in airway disease.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Músculo Liso/efectos de los fármacos , Tráquea/efectos de los fármacos , 16,16-Dimetilprostaglandina E2/farmacología , Animales , Western Blotting , Bovinos , Línea Celular , Activación Enzimática , Técnicas In Vitro , Músculo Liso/enzimología , Músculo Liso/fisiología , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Tráquea/enzimología , Tráquea/fisiología
17.
Respir Res ; 10: 88, 2009 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-19788733

RESUMEN

BACKGROUND: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. METHODS: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. RESULTS: The beta2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac. CONCLUSION: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.


Asunto(s)
Bradiquinina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Interleucina-8/metabolismo , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismo , Línea Celular , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos
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