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Electrospun drug-eluting fibers have demonstrated potentials in topical drug delivery applications, where drug releases can be modulated by polymer fiber compositions. In this study, blend fibers of polycaprolactone (PCL) and polyethylene oxide (PEO) at various compositions were electrospun from 10 wt% of polymer solutions to encapsulate a model drug of ibuprofen (IBP). The results showed that the average polymer solution viscosities determined the electrospinning parameters and the resulting average fiber diameters. Increasing PEO contents in the blend PCL/PEO fibers decreased the average elastic moduli, the average tensile strength, and the average fracture strains, where IBP exhibited a plasticizing effect in the blend PCL/PEO fibers. Increasing PEO contents in the blend PCL/PEO fibers promoted the surface wettability of the fibers. The in vitro release of IBP suggested a transition from a gradual release to a fast release when increasing PEO contents in the blend PCL/PEO fibers up to 120 min. The in vitro viability of blend PCL/PEO fibers using MTT assays showed that the fibers were compatible with MEF-3T3 fibroblasts. In conclusion, our results explained the scientific correlations between the solution properties and the physicomechanical properties of electrospun fibers. These blend PCL/PEO fibers, having the ability to modulate IBP release, are suitable for topical drug delivery applications.
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The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.
Assuntos
Mycobacterium tuberculosis , Terapia por Fagos , Tuberculose , Animais , Camundongos , Humanos , Tuberculose/terapia , Tuberculose/microbiologia , Macrófagos/microbiologiaRESUMO
During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-ß induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-ß stimulation at nucleus, suggesting that TGF-ß facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.
Assuntos
Fibronectinas , Proteínas Nucleares , Fator de Resposta Sérica , Transativadores , Humanos , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Fibronectinas/genética , Fatores de Transcrição , Fator de Crescimento Transformador beta/metabolismo , Colágeno , FibroseRESUMO
During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.
Assuntos
Miofibroblastos , Doenças Pleurais , Camundongos , Animais , Humanos , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Miofibroblastos/metabolismo , Fosforilação , Cadeias Leves de Miosina/metabolismo , Doenças Pleurais/metabolismo , Miosina Tipo II/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , FibroseRESUMO
Progressive lung scarring because of persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin and matrix proteins, including collagen-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs) (1). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mTOR complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is, likewise, a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors such as INK128 and AZD8055 significantly inhibited transforming growth factor ß (TGF-ß)-induced MesoMT markers in HPMCs. We further identified the mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-ß. Knockdown of Rictor, but not Raptor, attenuated TGF-ß-induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-ß-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae-mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in subacute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 is critical for MesoMT induction and that targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.
Assuntos
Doenças Pleurais , Pleurisia , Animais , Camundongos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Fatores de Transcrição , Fator de Crescimento Transformador beta/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , FibroseRESUMO
Fascin, a major actin cross-linking protein, is expressed in most vertebrate epithelial tissues. It organizes actin filaments into well-ordered bundles that are responsible for the extension of dynamic membrane protrusions, including microspikes, filopodia, and invadopodia from cell surfaces, which are involved in cell migration and invasion as critical components of cancer metastasis. However, it is not well-understood how fascin-1 induces actin binding/bundling and where fascin-1 localizes along the actin filaments, thus facilitating actin bundle formation. In the present study, we attempted to clarify these problems by using biochemical and electron microscopic analyses using various fascin-1 constructs. Three dimensional structures of actin/fascin-1 complex were obtained by electron microscopy (EM) with iterative helical real-space reconstruction (IHRSR) and tomography. We revealed that the N-terminal region containing the Actin-Binding Site 2 (ABS2) of fascin-1 is responsible for actin bundling and the C-terminal region is important for the dimerization of fascin-1. We also found that the dimerization of fascin-1 through intermolecular interactions of the C-terminal region is essential for actin bundling. Since fascin is an important factor in cancer development, it is expected that the findings of present study will provide useful information for development of therapeutic strategies for cancer.
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Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-ß significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-ß also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis.
Assuntos
Fibronectinas , Miosina Tipo V , Fibrose , Guanosina Trifosfato , Humanos , Cadeias Pesadas de Miosina , Miosinas , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of â¼0.4 µm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of â¼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail-tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.
Assuntos
Actinas , Miosinas , Citoesqueleto de Actina , Actinas/metabolismo , Mitocôndrias , Dinâmica Mitocondrial , Miosinas/metabolismo , Pseudópodes/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor ß (TGFß)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFß-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFß-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFß-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFß (Ad-TGFß)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Animais , Bleomicina , Caveolina 1/genética , Caveolina 1/metabolismo , Endorribonucleases/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Camundongos , Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases , Fator de Crescimento Transformador beta/metabolismoRESUMO
Programmed death ligand-1 (PD-L1) is an immune checkpoint protein that has been linked with idiopathic pulmonary fibrosis (IPF) and fibroblast to myofibroblast transition (FMT). However, it remains largely unclear how PD-L1 mediates this process. We found significantly increased PD-L1 in the lungs of idiopathic pulmonary fibrosis patients and mice with pulmonary fibrosis induced by bleomycin and TGF-ß. In primary human lung fibroblasts (HLFs), TGF-ß induced PD-L1 expression that is dependent on both Smad3 and p38 pathways. PD-L1 knockdown using siRNA significantly attenuated TGF-ß-induced expression of myofibroblast markers α-SMA, collagen-1, and fibronectin in normal and IPF HLFs. Further, we found that PD-L1 interacts with Smad3, and TGF-ß induces their interaction. Interestingly, PD-L1 knockdown reduced α-SMA reporter activity induced by TGF-ß in HLFs, suggesting that PD-L1 might act as a co-factor of Smad3 to promote target gene expression. TGF-ß treatment also phosphorylates GSK3ß and upregulates ß-catenin protein levels. Inhibiting ß-catenin signaling with the pharmaceutical inhibitor ICG001 significantly attenuated TGF-ß-induced FMT. PD-L1 knockdown also attenuated TGF-ß-induced GSK3ß phosphorylation/inhibition and ß-catenin upregulation, implicating GSK3ß/ß-catenin signaling in PD-L1-mediated FMT. Collectively, our findings demonstrate that fibroblast PD-L1 may promote pulmonary fibrosis through both Smad3 and ß-catenin signaling and may represent a novel interventional target for IPF.
Assuntos
Antígeno B7-H1/metabolismo , Proteína Smad3/metabolismo , beta Catenina/metabolismo , Idoso , Animais , Antígeno B7-H1/genética , Bleomicina/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. Transforming growth factor-ß (TGF-ß) induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain α-smooth muscle actin (αSMA) associated with calponin 1 but not ß-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-ß significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis (PF) and that upregulation of calponin 1 plays a central role in this process.
Assuntos
Miofibroblastos , Pleura , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Células Cultivadas , Fibrose , Humanos , Proteínas dos Microfilamentos , Miofibroblastos/metabolismo , Pleura/patologia , Fator de Crescimento Transformador beta/farmacologia , CalponinasRESUMO
Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-ß [transforming growth factor-ß], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-ß. TGF-ß significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-ß-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-ß-induced MesoMT. DOCK2 knockdown also inhibited TGF-ß-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.
Assuntos
Transição Epitelial-Mesenquimal , Epitélio/patologia , Fibrose/patologia , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Pleura/patologia , Pleurisia/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Modelos Animais de Doenças , Epitélio/metabolismo , Fibrose/induzido quimicamente , Fibrose/metabolismo , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pleura/metabolismo , Pleurisia/induzido quimicamente , Pleurisia/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genéticaRESUMO
Pleural fibrosis (PF) is a chronic and progressive lung disease which affects approximately 30,000 people per year in the United States. Injury and sustained inflammation of the pleural space can result in PF, restricting lung expansion and impairing oxygen exchange. During the progression of pleural injury, normal pleural mesothelial cells (PMCs) undergo a transition, termed mesothelial mesenchymal transition (MesoMT). While multiple components of the fibrinolytic pathway have been investigated in pleural remodeling and PF, the role of the urokinase type plasminogen activator receptor (uPAR) is unknown. We found that uPAR is robustly expressed by pleural mesothelial cells in PF. Downregulation of uPAR by siRNA blocked TGF-ß mediated MesoMT. TGF-ß was also found to significantly induce uPA expression in PMCs undergoing MesoMT. Like uPAR, uPA downregulation blocked TGF-ß mediated MesoMT. Further, uPAR is critical for uPA mediated MesoMT. LRP1 downregulation likewise blunted TGF-ß mediated MesoMT. These findings are consistent with in vivo analyses, which showed that uPAR knockout mice were protected from S. pneumoniae-mediated decrements in lung function and restriction. Histological assessments of pleural fibrosis including pleural thickening and α-SMA expression were likewise reduced in uPAR knockout mice compared to WT mice. These studies strongly support the concept that uPAR targeting strategies could be beneficial for the treatment of PF.
Assuntos
Transição Epitelial-Mesenquimal , Pneumonia Bacteriana/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Infecções Estreptocócicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Epitélio/metabolismo , Epitélio/patologia , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pleura/metabolismo , Pleura/patologia , Pneumonia Bacteriana/patologia , Infecções Estreptocócicas/patologia , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.
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Transição Epitelial-Mesenquimal , NADPH Oxidase 1/metabolismo , Pleura/enzimologia , Pleurisia/enzimologia , Pneumonia Pneumocócica/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/patogenicidade , Animais , Células Cultivadas , Modelos Animais de Doenças , Fator Xa/metabolismo , Fibrose , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1/deficiência , NADPH Oxidase 1/genética , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Pleura/microbiologia , Pleura/patologia , Pleurisia/microbiologia , Pleurisia/patologia , Pleurisia/fisiopatologia , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Transdução de Sinais , Trombina/metabolismoRESUMO
Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9-12, which can be activated to form functional filaments as needed13-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.
Assuntos
Microscopia Crioeletrônica , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/ultraestrutura , Animais , Sítios de Ligação , Modelos Moleculares , Músculo Liso/química , Mutação , Miosina Tipo II/química , Miosina Tipo II/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Desdobramento de Proteína , PerusRESUMO
Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Miócitos de Músculo Liso/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Colforsina/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Histamina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatase de Miosina-de-Cadeia-Leve/genética , Adulto JovemRESUMO
Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, ß, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR. Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells. Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .
Assuntos
Canais Epiteliais de Sódio/genética , Modelos Animais , Células Epiteliais Alveolares/metabolismo , Animais , Canais Epiteliais de Sódio/metabolismo , Expressão Gênica , Humanos , Transporte de Íons/genética , Transporte de Íons/fisiologia , Camundongos , Camundongos Transgênicos/metabolismo , Oócitos , Células-Tronco/metabolismo , XenopusRESUMO
Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.
Assuntos
Músculo Liso/metabolismo , Miosinas de Músculo Liso/química , Miosinas de Músculo Liso/metabolismo , Animais , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , PerusRESUMO
Pleural fibrosis is characterized by severe inflammation of the pleural space and pleural reorganization. Subsequent thickening of the visceral pleura contributes to lung stiffness and impaired lung function. Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, the mechanisms that underlie MesoMT remain unclear. Here, we investigated the role of myocardin in the induction of MesoMT. Transforming growth factor ß (TGF-ß) and thrombin induced MesoMT and markedly upregulated the expression of myocardin, but not myocardin-related transcription factor A (MRTF-A) or MRTF-B, in human PMCs (HPMCs). TGF-ß stimulation notably induced the nuclear translocation of myocardin in HPMCs, whereas nuclear translocation of MRTF-A and MRTF-B was not observed. Several genes under the control of myocardin were upregulated in cells undergoing MesoMT, an effect that was accompanied by a dramatic cytoskeletal reorganization of HPMCs consistent with a migratory phenotype. Myocardin gene silencing blocked TGF-ß- and thrombin-induced MesoMT. Although myocardin upregulation was blocked, MRTF-A and MRTF-B were unchanged. Myocardin, α-SMA, calponin, and smooth muscle myosin were notably upregulated in the thickened pleura of carbon black/bleomycin and empyema mouse models of fibrosing pleural injury. Similar results were observed in human nonspecific pleuritis. In a TGF-ß mouse model of pleural fibrosis, PMC-specific knockout of myocardin protected against decrements in lung function. Further, TGF-ß-induced pleural thickening was abolished by PMC-specific myocardin knockout, which was accompanied by a marked reduction of myocardin, calponin, and α-SMA expression compared with floxed-myocardin controls. These novel results show that myocardin participates in the development of MesoMT in HPMCs and contributes to the pathogenesis of pleural organization and fibrosis.
Assuntos
Núcleo Celular/metabolismo , Empiema Pleural/metabolismo , Miofibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Pleura/metabolismo , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Núcleo Celular/patologia , Modelos Animais de Doenças , Empiema Pleural/induzido quimicamente , Empiema Pleural/patologia , Feminino , Fibrose , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Miofibroblastos/patologia , Pleura/patologia , Fuligem/toxicidade , Fator de Crescimento Transformador beta/metabolismoRESUMO
Myosin X is an unconventional actin-based molecular motor involved in filopodial formation, microtubule-actin filament interaction, and cell migration. Myosin X is an important component of filopodia regulation, localizing to tips of growing filopodia by an unclear targeting mechanism. The native α-helical dimerization domain of myosin X is thought to associate with antiparallel polarity of the two amino acid chains, making myosin X the only myosin that is currently considered to form antiparallel dimers. This study aims to determine if antiparallel dimerization of myosin X imparts selectivity toward actin bundles by comparing the motility of parallel and antiparallel dimers of myosin X on single and fascin-bundled actin filaments. Antiparallel myosin X dimers exhibit selective processivity on fascin-bundled actin and are only weakly processive on single actin filaments below saturating [ATP]. Artificial forced parallel dimers of myosin X are robustly processive on both single and bundled actin, exhibiting no selectivity. To determine the relationship between gating of the reaction steps and observed differences in motility, a mathematical model was developed to correlate the parameters of motility with the biochemical and mechanical kinetics of the dimer. Results from the model, constrained by experimental data, suggest that the probability of binding forward, toward the barbed end of the actin filament, is lower in antiparallel myosin X on single actin filaments compared to fascin-actin bundles and compared to constructs of myosin X with parallel dimerization.