RESUMO
Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.
Assuntos
Hipertensão Pulmonar , Quinase I-kappa B , Hipertensão Arterial Pulmonar , Remodelação Vascular , Animais , Humanos , Ratos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Quinase I-kappa B/metabolismo , Miócitos de Músculo Liso , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar , Proteínas de Sinalização YAP/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismoRESUMO
Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor ß1 (TGFß1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.
Assuntos
Fibroblastos/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/metabolismo , Fenômenos Biomecânicos/efeitos dos fármacos , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Yes-associated protein (YAP) and PDZ-binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs-coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2-mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.
Assuntos
Fibroblastos/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Dopamina D1/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas ras/metabolismo , Células Cultivadas , Agonistas de Dopamina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Fenantridinas/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Dopamina D1/agonistas , Transdução de Sinais , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas de Sinalização YAP/genética , Proteínas ras/genéticaRESUMO
Injuries caused by mobile machinery or special purpose vehicles (SPVs) can lead to high socio-medical cost and fatality. In this descriptive study, we compared the epidemiology and injury profile of injured patients involved in SPVs-related incidents. We analyzed a nationwide database of SPV-related injured patients between January 2011 and December 2016. Injured patients were classified into three groups: pedestrian, motor vehicle occupant (MVO), and SPV operator groups. Of 1,419 cases, the highest number of SPV-related injured patients were found in the age group 40-59 years (671 cases, 47.3%) and at transport area (771 cases, 54.3%). The injury was most severe in the SPV operator group. The lower extremities were the most common fracture site, and intrathoracic injury was the most common visceral regions for SPV-related injured patients. SPV operator could lead to fatal intrathoracic injuries.
Assuntos
Acidentes de Trânsito/estatística & dados numéricos , Veículos Automotores/classificação , Veículos Automotores/estatística & dados numéricos , Ferimentos e Lesões/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Saúde Ocupacional , República da Coreia/epidemiologia , Fatores de Risco , Fatores Sexuais , Índices de Gravidade do Trauma , Caminhada , Adulto JovemRESUMO
Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Fatores de Transcrição/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Comunicação Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fator de Crescimento Transformador beta/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAPRESUMO
Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Fibroblastos/patologia , Cirrose Hepática/patologia , Fibrose Pulmonar/patologia , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Transativadores/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bleomicina , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dopa Descarboxilase/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Lesão Pulmonar/patologia , Masculino , Camundongos Endogâmicos C57BL , Fenantridinas/farmacologia , Fenótipo , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Transativadores/metabolismo , Proteínas de Sinalização YAPRESUMO
Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFß and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFß- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.
Assuntos
Proteínas Cromossômicas não Histona/genética , Fibroblastos/metabolismo , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Fibrose Pulmonar Idiopática/genética , Pulmão/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Fibroblastos/patologia , Inativação Gênica , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.
Assuntos
Janus Quinase 2/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/genética , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Feminino , Fibroblastos/metabolismo , Humanos , Janus Quinase 2/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fibrose Pulmonar/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Quinases Associadas a rho/genéticaRESUMO
Lung function is inherently mechanical in nature and depends on the capacity to conduct air and blood to and from the gas exchange regions. Variations in the elastic properties of the human lung across anatomical compartments and with aging are likely important determinants of lung function but remain relatively poorly characterized. Here we applied atomic force microscopy microindentation to characterize human lung tissue from subjects ranging in age from 11 to 60 yr old. We observed striking anatomical variations in elastic modulus, with the airways (200- to 350-µm diameter) the stiffest and the parenchymal regions the most compliant. Vessels (diameter < 100 µm) represented an intermediate mechanical environment and displayed diameter-dependent trends in elastic modulus. Binning our samples into younger (11-30 yr old) and older (41-60 yr old) groups, we observed significant age-related increases in stiffness in parenchymal and vessel compartments, with the most pronounced changes in the vessels. To investigate cellular mechanisms that might contribute to vascular stiffening with aging, we studied primary human pulmonary artery smooth muscle cells from subjects ranging in age from 11 to 60 yr old. While we observed no change in the mechanical properties of the cells themselves, we did observe trends toward increases in traction forces and extracellular matrix deposition with aging. These results demonstrate age-related changes in tissue mechanical properties that likely contribute to impaired lung function with aging and underscore the potential to identify mechanisms that contribute to mechanical tissue remodeling through the study of human cells and tissues from across the aging spectrum.
Assuntos
Envelhecimento/metabolismo , Remodelação das Vias Aéreas , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Pulmão/metabolismo , Pulmão/ultraestrutura , Adolescente , Adulto , Criança , Módulo de Elasticidade , Feminino , Humanos , Masculino , Microscopia de Força Atômica , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Idiopathic and diabetic gastroparesis in Homo sapiens cause significant morbidity. Etiology or risk factors have not been clearly identified. Failure to sustain elevated heme oxygenase-1 (HO1) expression is associated with delayed gastric emptying in diabetic mice and polymorphisms in the HO1 gene (HMOX1, NCBI Gene ID:3162) are associated with worse outcomes in other diseases. AIM: Our hypothesis was that longer polyGT alleles are more common in the HMOX1 genes of individuals with gastroparesis than in controls without upper gastrointestinal motility disorders. METHODS: Repeat length was determined in genomic DNA. Controls with diabetes (84 type 1, 84 type 2) and without diabetes (n = 170) were compared to diabetic gastroparetics (99 type 1, 72 type 2) and idiopathic gastroparetics (n = 234). Correlations of repeat lengths with clinical symptom sub-scores on the gastroparesis cardinal symptom index (GCSI) were done. Statistical analyses of short (<29), medium and long (>32) repeat alleles and differences in allele length were used to test for associations with gastroparesis. RESULTS: The distribution of allele lengths was different between groups (P = 0.016). Allele lengths were longest in type 2 diabetics with gastroparesis (29.18±0.35, mean ± SEM) and longer in gastroparetics compared to non-diabetic controls (28.50±0.14 vs 27.64±0.20 GT repeats/allele, P = 0.0008). Type 2 diabetic controls had longer alleles than non-diabetic controls. In all gastroparetic groups, allele lengths were longer in African Americans compared to other racial groups, differences in the proportion of African Americans in the groups accounted for the differences between gastroparetics and controls. Diabetic gastroparetics with 1 or 2 long alleles had worse GCSI nausea sub-scores (3.30±0.23) as compared to those with 0 long alleles (2.66±0.12), P = 0.022. CONCLUSIONS: Longer poly-GT repeats in the HMOX1 gene are more common in African Americans with gastroparesis. Nausea symptoms are worse in subjects with longer alleles.
Assuntos
Diabetes Mellitus/genética , Gastroparesia/genética , Heme Oxigenase-1/genética , Sequências de Repetição em Tandem/genética , Adulto , Negro ou Afro-Americano/genética , Idoso , Alelos , Animais , Complicações do Diabetes/genética , Complicações do Diabetes/patologia , Diabetes Mellitus/patologia , Feminino , Esvaziamento Gástrico/genética , Gastroparesia/patologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo GenéticoRESUMO
Pulmonary arterial stiffness is an independent risk factor for mortality in pulmonary hypertension (PH) and plays a critical role in PH pathophysiology. Our laboratory has recently demonstrated arterial stiffening early in experimental PH, along with evidence for a mechanobiological feedback loop by which arterial stiffening promotes further cellular remodeling behaviors (Liu F, Haeger CM, Dieffenbach PB, Sicard D, Chrobak I, Coronata AM, Suárez Velandia MM, Vitali S, Colas RA, Norris PC, Marinkovic A, Liu X, Ma J, Rose CD, Lee SJ, Comhair SA, Erzurum SC, McDonald JD, Serhan CN, Walsh SR, Tschumperlin DJ, Fredenburgh LE. JCI Insight 1: e86987, 2016). Cyclooxygenase-2 (COX-2) and prostaglandin signaling have been implicated in stiffness-mediated regulation, with prostaglandin activity inversely correlated to matrix stiffness and remodeling behaviors in vitro, as well as to disease progression in rodent PH models. The mechanism by which mechanical signaling translates to reduced COX-2 activity in pulmonary vascular cells is unknown. The present work investigated the transcriptional regulators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1, a.k.a., TAZ), which are known drivers of downstream mechanical signaling, in mediating stiffness-induced changes in COX-2 and prostaglandin activity in pulmonary artery smooth muscle cells (PASMCs). We found that YAP/TAZ activity is increased in PAH PASMCs and experimental PH and is necessary for the development of stiffness-dependent remodeling phenotypes. Knockdown of YAP and TAZ markedly induces COX-2 expression and downstream prostaglandin production by approximately threefold, whereas overexpression of YAP or TAZ reduces COX-2 expression and prostaglandin production to near undetectable levels. Together, our findings demonstrate a stiffness-dependent YAP/TAZ-mediated positive feedback loop that drives remodeling phenotypes in PASMCs via reduced COX-2 and prostaglandin activity. The ability to interrupt this critical mechanobiological feedback loop and enhance local prostaglandin activity via manipulation of YAP/TAZ signaling presents a highly attractive novel strategy for the treatment of PH.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Remodelação das Vias Aéreas/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo-Oxigenase 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Rigidez Vascular/fisiologia , Adulto , Animais , Movimento Celular , Proliferação de Células , Demografia , Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hipertensão Pulmonar , Masculino , Pessoa de Meia-Idade , Fenótipo , Artéria Pulmonar/citologia , Ratos Sprague-Dawley , Transdução de Sinais , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Mechanical ventilation is necessary to support patients with acute lung injury, but also exacerbates injury through mechanical stress-activated signaling pathways. We show that stretch applied to cultured human cells, and to mouse lungs in vivo, induces robust expression of metallothionein, a potent antioxidant and cytoprotective molecule critical for cellular zinc homeostasis. Furthermore, genetic deficiency of murine metallothionein genes exacerbated lung injury caused by high tidal volume mechanical ventilation, identifying an adaptive role for these genes in limiting lung injury. Stretch induction of metallothionein required zinc and the zinc-binding transcription factor MTF1. We further show that mouse dietary zinc deficiency potentiates ventilator-induced lung injury, and that plasma zinc levels are significantly reduced in human patients who go on to develop acute respiratory distress syndrome (ARDS) compared with healthy and non-ARDS intensive care unit (ICU) controls, as well as with other ICU patients without ARDS. Taken together, our findings identify a potentially novel adaptive response of the lung to stretch and a critical role for zinc in defining the lung's tolerance for mechanical ventilation. These results demonstrate that failure of stretch-adaptive responses play an important role in exacerbating mechanical ventilator-induced lung injury, and identify zinc and metallothionein as targets for lung-protective interventions in patients requiring mechanical ventilation.
RESUMO
BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Esvaziamento Gástrico/fisiologia , Hiperglicemia/fisiopatologia , Células Intersticiais de Cajal/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fatores de Transcrição/fisiologia , Animais , Feminino , Humanos , Células Intersticiais de Cajal/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Receptores para Leptina/genética , Regulação para CimaRESUMO
Recent studies have implicated the Hippo pathway and its transcriptional effectors YAP and TAZ as necessary for fibroblast activation and tissue fibrosis. To test the specific and sufficient roles for TAZ in driving autonomous fibroblast activation, we cultured NIH3T3 fibroblasts expressing a doxycycline-inducible nuclear-localized mutant of TAZ (TAZ4SA) in scaffold-free 3D hanging drop spheroids, or on matrices of specified mechanical rigidity. Control NIH3T3 fibroblasts formed spheroids in hanging drop culture that remained stable and neither increased nor decreased in size significantly over 15 days. In contrast, TAZ4SA-transduced fibroblasts grew robustly in spheroid culture, and expressed enhanced levels of genes encoding profibrotic soluble factors connective tissue growth factor (CTGF), endothelin-1 (Et-1), and plasminogen activator inhibitor 1 (PAI-1). However, TAZ4SA expression was unable to enhance expression of extracellular matrix (ECM)-encoding genes Col1a1, Col1a2, Col3a1, or Fn1 in spheroid culture. Micromechanical testing indicated that spheroids composed of either control or TAZ4SA-expressing cells were highly compliant and indistinguishable in mechanical properties. In fibroblasts cultured on 2D matrices of compliance similar to spheroids, TAZ4SA expression was able to enhance contractile force generation, but was unable to enhance ECM gene expression. In contrast, culture on stiff hydrogels potentiated TAZ4SA enhancement of ECM expression. TAZ4SA enhancement of Col1a1 expression on soft matrices was potentiated by TGF-ß1, while on stiff matrices it was abrogated by inhibition of myocardin-related transcription factor, demonstrating context-dependent crosstalk of TAZ with these pathways. These findings demonstrate sufficiency of TAZ activation for driving fibroblast proliferation, contraction, and soluble profibrotic factor expression, and mechanical context-dependent crosstalk of TAZ with other pathways in regulating Col1a1 expression.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Comunicação Parácrina , Esferoides Celulares/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Proliferação de Células , Células Cultivadas , Fibroblastos/patologia , Fibrose , Camundongos , Células NIH 3T3 , Esferoides Celulares/patologia , Ativação TranscricionalRESUMO
Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-ß1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.
Assuntos
Pulmão/citologia , Pulmão/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Engenharia Tecidual/métodos , Adulto , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Epiteliais/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Organogênese , Organoides/transplante , RegeneraçãoRESUMO
BACKGROUND & AIMS: Gastroparesis is a complication of diabetes characterized by delayed emptying of stomach contents and accompanied by early satiety, nausea, vomiting, and pain. No safe and reliable treatments are available. Interleukin 10 (IL10) activates the M2 cytoprotective phenotype of macrophages and induces expression of heme oxygenase 1 (HO1) protein. We investigated whether IL10 administration could improve gastric emptying and reverse the associated cellular and electrical abnormalities in diabetic mice. METHODS: Nonobese diabetic mice with delayed gastric emptying were given either IL10 (0.1-1 µg, twice/day) or vehicle (controls). Stomach tissues were isolated, and sharp microelectrode recordings were made of the electrical activity in the gastric muscle layers. Changes to interstitial cells of Cajal (ICC), reduced nicotinamide adenine dinucleotide phosphate diaphorase, and levels and distribution of HO1 protein were determined by histochemical and imaging analyses of the same tissues. RESULTS: Gastric emptying remained delayed in vehicle-treated diabetic mice but returned to normal in mice given IL10 (n = 10 mice; P < .05). In mice given IL10, normalization of gastric emptying was associated with a membrane potential difference between the proximal and distal stomach, and lower irregularity and higher frequency of slow-wave activity, particularly in the distal stomach. Levels of HO1 protein were higher in stomach tissues from mice given IL10, and ICC networks were more organized, better connected, and more evenly distributed compared with controls. CONCLUSIONS: IL10 increases gastric emptying in diabetic mice and has therapeutic potential for patients with diabetic gastroparesis. This response is associated with up-regulation of HO1 and repair of connectivity of ICC networks.
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BACKGROUND & AIMS: Diabetic gastroparesis is associated with changes in interstitial cells of Cajal (ICC), neurons and smooth muscle cells in both animal models and humans. Macrophages appear to be critical to the development of cellular damage that leads to delayed gastric emptying but the mechanisms involved are not well understood. Csf1op/op (Op/Op) mice lack biologically active Csf1, resulting in the absence of Csf1-dependent tissue macrophages. The aim of this study was to use Csf1op/op mice to determine the role of macrophages in the development of delayed gastric emptying. METHODS: Animals were injected with streptozotocin to make them diabetic. Gastric emptying was determined weekly. Immunohistochemistry was used to identify macrophages and ICC networks in the gastric muscular layers. Oxidative stress was measured by serum malondialdehyde (MDA) levels. Quantitative, reverse transcription PCR was used to measure levels of mRNA. RESULTS: Csf1op/op mice had normal ICC. With onset of diabetes both Csf1op/op and wild type Csf1+/+ mice developed increased levels of oxidative stress (75.8 ± 9.1 and 41.2±13.6 nmol/mL MDA respectively). Wild type Csf1+/+ mice developed delayed gastric emptying after onset of diabetes (4/13) whereas no diabetic Csf1op/op mouse developed delayed gastric emptying (0/15, P=0.035). ICC were disrupted in diabetic wild type Csf1+/+ mice with delayed gastric emptying but remained normal in diabetic Csf1op/op mice. CONCLUSIONS: Cellular injury and development of delayed gastric emptying in diabetes requires the presence of muscle layer macrophages. Targeting macrophages may be an effective therapeutic option to prevent cellular damage and development of delayed gastric emptying in diabetes.
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BACKGROUND & AIMS: In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. METHODS: We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. RESULTS: KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. CONCLUSIONS: In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.
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Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Benzamidas/metabolismo , Benzimidazóis/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Tumores do Estroma Gastrointestinal/genética , Técnicas de Silenciamento de Genes/métodos , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Precursores de Ácido Nucleico/genética , Fosforilação/genética , Piperazinas/metabolismo , Piperidinas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-ß signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/metabolismo , Fosfoproteínas/metabolismo , Fibrose Pulmonar/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Feminino , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/patologia , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos NOD , Mutação , Células NIH 3T3 , Fosfoproteínas/genética , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Serpina E2/biossíntese , Serpina E2/genética , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Heme oxygenase 1 (HO-1) degrades heme and protects against oxidative stress. In vitro and animal models suggest that HO-1 is beneficial in several diseases (e.g., postoperative ileus, gastroparesis, acute pancreatitis, and colitis). However, the only drugs (i.e., hemin and heme arginate) which pharmacologically upregulate HO-1 in humans are expensive and can only be administered intravenously. Our aims were to compare the effects of placebo, aspirin, and simvastatin alone, and with α-lipoic acid, on HO-1 protein concentration and activity in humans. METHODS: This randomized, double-blind, placebo-controlled study compared the effects of three oral regimens administered for 7 days, i.e., placebo; aspirin (325 mg twice daily) and simvastatin (40 mg twice daily); aspirin, simvastatin, and the sodium salt of R- α-lipoic acid (NaRLA, 600 mg three times daily) on markers of HO-1 activation (i.e., plasma HO-1 protein concentration and venous monocyte HO-1 protein activity) in 18 healthy subjects (14 females). Markers of HO-1 activation were evaluated at baseline, days 2, and 7. KEY RESULTS: Baseline HO-1 protein concentrations and activity were similar among the three groups. Compared to placebo, aspirin and simvastatin combined, or together with NaRLA did not affect HO-1 protein concentration or activity at 2 or 7 days. HO-1 protein concentrations and activity were correlated on day 7 (r = 0.75, p = 0.0004) but not at baseline and on day 2. CONCLUSIONS & INFERENCES: At therapeutic doses, aspirin, simvastatin, and α-lipoic acid do not increase plasma HO-1 protein concentration or venous monocyte HO-1 activity in healthy humans.