RESUMEN
Platelet-derived growth factor receptor-α (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRα and Nrp1 colocalized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live-cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRα, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRα co-immunoprecipitated with APPL1, indicating that PDGFRα enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.
Asunto(s)
Endosomas/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Neuropilina-1/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Endosomas/genética , Fibroblastos/citología , Pulmón/citología , Ratones , Ratones Noqueados , Neuropilina-1/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
During pulmonary secondary alveolar septation, the rudimentary distal saccule subdivides by extending tissue sheets into the saccular air space, creating alveoli, which open into the alveolar duct. The sheets originate from saccular mesenchymal cells, which contain α-SMA (αSMA [ACTA2]) and abut elastic fibers (myofibroblasts [MF]), characteristics that are shared by cells that subsequently occupy the secondary septal tips. During elongation, collagen fibers are positioned to provide a scaffold for translocating septal mesenchymal cells. We hypothesized that collagen fibers direct the migration, orientation, and location of MFs during septal elongation. To address this hypothesis, we examined how electrospun collagen fibers direct the migration of fibroblasts bearing targeted deletions of PDGFRα (platelet-derived growth factor receptor-α) or Nrp1 (neuropilin-1), after their isolation from lungs that exhibit reduced secondary septation. We observed that deletion of either gene reduced Rac1 activation and the speed of migration of lung fibroblasts (LF) along electrospun fibers. The deletions did not reduce the proportion of LF that displayed collagen-binding integrins and increased the proportion of LF bearing activated ß1-integrin. LF bearing the PDGFRα deletion failed to localize focal adhesions over electrospun fibers, suggesting that they may not appropriately sense and respond to regionally increased stiffness near the fibers. In lungs of mice bearing the PDGFRα deletion, collagen fibers are delocalized from ACTA2-containing MF, and their orientation deviated from the plane of the alveolar walls. Diminished PDGFRα or Nrp1 reduces LF localization to stiffer regions of fibrillar collagen substrates, suggesting that signaling through these receptors enables responsiveness to regional differences in extracellular matrix rigidity.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Miofibroblastos/metabolismo , Neuropilina-1/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Actinas/metabolismo , Animales , Matriz Extracelular/metabolismo , Femenino , Adhesiones Focales/metabolismo , Masculino , Ratones , Alveolos Pulmonares/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Generation of secondary alveolar septa occurs primarily after birth in humans and is complete in mice postnatally, when mechanical stresses vary as air space pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers, which limit cell strain; although when the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cell division and migration, which occur during septal elongation. Because platelet-derived growth factor receptor-α (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1 gene deletion in alveolar mesenchymal cells (myofibroblasts and pericytes). NRP1 depletion reduced the proportion of mesenchymal cells that contain nestin and desmin within the subpopulation that lacked PDGFRα but contained PDGFRß. Desmin was reduced at alveolar entry rings, air spaces were enlarged, and surface area was reduced after NRP1 depletion. PDGFRα and NRP1 colocalized to membrane lipid rafts, which are known to contain Src kinase. NRP1 depletion reduced alveolar mesenchymal cell migration and PDGF-A-mediated activation of Src kinase, which may limit accumulation of desmin at septal tips (alveolar entry rings). Cooperation between NRP1 and PDGF signaling is required for secondary septation, and manipulation of NRP1 could promote alveolar regeneration without producing fibrosis.
Asunto(s)
Filamentos Intermedios/metabolismo , Neuropilina-1/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Alveolos Pulmonares/crecimiento & desarrollo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Animales , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Eliminación de Gen , Filamentos Intermedios/genética , Ratones , Ratones Transgénicos , Neuropilina-1/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Glucocorticoids have been widely used and exert pleiotropic effects on alveolar structure and function, but do not improve the long-term clinical outcomes for patients with bronchopulmonary dysplasia, emphysema, or interstitial lung diseases. Treatments that foster alveolar regeneration could substantially improve the long-term outcomes for such patients. One approach to alveolar regeneration is to stimulate and guide intrinsic alveolar progenitors along developmental pathways used during secondary septation. Other investigators and we have identified platelet-derived growth factor receptor-α-expressing fibroblast subpopulations that are alternatively skewed toward myofibroblast or lipofibroblast phenotypes. In this study, we administered either the glucocorticoid receptor agonist dexamethasone (Dex) or the antagonist mifepristone to mice during the first postnatal week and evaluated their effects on cellular proliferation and adoption of α-smooth muscle actin and lipid droplets (markers of the myofibroblast and lipofibroblast phenotypes, respectively). We observed that Dex increased the relative abundance of fibroblasts with progenitor characteristics, i.e., containing both α-smooth muscle actin and lipid droplets, uncoupling protein-1 (a marker of brown and beige adipocytes), delta-like ligand-1, and stem cell antigen-1. Dex enhanced signaling through the Smad1/5 pathway, which increased uncoupling protein-1 in a lung fibroblast progenitor cell line. We conclude that glucocorticoid receptor manipulation can sustain fibroblast plasticity, and posit that targeting downstream glucocorticoid responsive pathways could steer fibroblast progenitors along more desirable regenerative pathways.
Asunto(s)
Fibroblastos/citología , Glucocorticoides/farmacología , Alveolos Pulmonares/crecimiento & desarrollo , Células Madre/citología , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Mifepristona/farmacología , Fosforilación/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Proteína Desacopladora 1/metabolismoRESUMEN
Platelet-derived growth factor (PDGF)-A, which only signals through PDGF-receptor-α (PDGFR-α), is required for secondary alveolar septal formation. Although PDGFR-α distinguishes mesenchymal progenitor cells during the saccular stage, PDGFR-α-expressing alveolar cells persist through adulthood. PDGF-A sustains proliferation, limits apoptosis, and maintains α-smooth muscle actin (α-SMA)-containing alveolar cells, which congregate at the alveolar entry ring at postnatal day (P)12. PDGFR-α-expressing, α-SMA-containing alveolar cells redistribute in the elongating septum, suggesting that they migrate to the alveolar entry rings, where mechanical tension is higher. We hypothesized that PDGFR-α and Ras-related C3 botulinum toxin substrate 1(Rac1) are required for mechanosensitive myofibroblast migration. Spreading of PDGFR-α-deficient lung fibroblasts was insensitive to increased rigidity, and their migration was not reduced by Rac1-guanine exchange factor (GEF)-inhibition. PDGFR-α-expressing fibroblasts migrated toward stiffer regions within two-dimensional substrates by increasing migrational persistence (durotaxis). Using a Förster resonance energy transfer (FRET) biosensor for Rac1-GTP, we observed that PDGFR-α was required for fibroblast Rac1 responsiveness to stiffness within a three-dimensional collagen substrate, which by itself increased Rac1-FRET. Rho-GTPase stabilized, whereas Rac1-GTPase increased the turnover of focal adhesions. Under conditions that increased Rac1-GTP, PDGFR-α signaled through both phosphoinositide-3-kinase (PIK) or Src to engage the Rac1 GEF dedicator of cytokinesis-1 (Dock180) and p21-activated-kinase interacting exchange factor-ß (ßPIX). In cooperation with collagen fibers, these signaling pathways may guide fibroblasts toward the more rigid alveolar entry ring during secondary septation. Because emphysema and interstitial fibrosis disrupt the parenchymal mechanical continuum, understanding how mechanical factors regulate fibroblast migration could elicit strategies for alveolar repair and regeneration.
Asunto(s)
Movimiento Celular , Fibroblastos/metabolismo , Mecanotransducción Celular , Neuropéptidos/metabolismo , Alveolos Pulmonares/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Fibroblastos/patología , Ratones , Ratones Noqueados , Neuropéptidos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Alveolos Pulmonares/patología , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genética , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Pulmonary alveolar fibroblasts produce extracellular matrix in a temporally and spatially regulated pattern to yield a durable yet pliable gas-exchange surface. Proliferation ensures a sufficient complement of cells, but they must differentiate into functionally distinct subtypes: contractile myofibroblasts (MF), which generate elastin and regulate air-flow at the alveolar ducts, and, in mice and rats, lipofibroblasts (LF), which store neutral lipids. PDGF-A is required but acts in conjunction with other differentiation factors arising from adjacent epithelia or within fibroblasts. We hypothesized that FGF receptor (FGFR) expression and function vary for MF and LF and contributes to their divergent differentiation. Whereas approximately half of the FGFR3 was extracellular in MF, FGFR2 and FGFR4 were primarily intracellular. Intracellular FGFR3 localized to the multivesicular body, and its abundance may be modified by Sprouty and interaction with heat shock protein-90. FGF18 mRNA is more abundant in MF, whereas FGF10 mRNA predominated in LF, which also express FGFR1 IIIb, a receptor for FGF10. FGF18 diminished fibroblast proliferation and was chemotactic for cultured fibroblasts. Although PDGF receptor-α (PDGFR-α) primarily signals through phosphoinositide 3-kinase and Akt, p42/p44 MAP kinase (Erk1/2), a major signaling pathway for FGFRs, influenced the abundance of cell-surface PDGFR-α. Observing different FGFR and ligand profiles in MF and LF is consistent with their divergent differentiation although both subpopulations express PDGFR-α. These studies also emphasize the importance of particular cellular locations of FGFR3 and PDGFR-α, which may modify their effects during alveolar development or repair.
Asunto(s)
Miofibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Alveolos Pulmonares/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Chaperonina 10/genética , Quinasas Ciclina-Dependientes , Elastina/biosíntesis , Matriz Extracelular/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Cuerpos Multivesiculares/metabolismo , Miofibroblastos/citología , Alveolos Pulmonares/citología , ARN Mensajero/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Signaling through platelet-derived growth factor receptor-α (PDGFRα) is required for alveolar septation and participates in alveolar regeneration after pneumonectomy. In both adipose tissue and skeletal muscle, bipotent pdgfrα-expressing progenitors expressing delta-like ligand-1 or sex-determining region Y box 9 (Sox9) may differentiate into either lipid storage cells or myofibroblasts. We analyzed markers of mesenchymal progenitors and differentiation in lung fibroblasts (LF) with different levels (absent, low, or high) of pdgfrα gene expression. A larger proportion of pdgfrα-expressing than nonexpressing LF contained Sox9. Neutral lipids, CD166, and Tcf21 were more abundant in LF with a lower compared with a higher level of pdgfrα gene expression. PDGF-A increased Sox9 in primary LF cultures, suggesting that active signaling through PDGFRα is required to maintain Sox9. As alveolar septation progresses from postnatal day (P) 8 to P12, fewer pdgfrα-expressing LF contain Sox9, whereas more of these LF contain myocardin-like transcription factor-A, showing that Sox9 diminishes as LF become myofibroblasts. At P8, neutral lipid droplets predominate in LF with the lower level of pdgfrα gene expression, whereas transgelin (tagln) was predominantly expressed in LF with higher pdgfrα gene expression. Targeted deletion of pdgfrα in LF, which expressed tagln, reduced Sox9 in α-actin (α-SMA, ACTA2)-containing LF, whereas it increased the abundance of cell surface delta-like protein-1 (as well as peroxisome proliferator-activated receptor-γ and tcf21 mRNA in LF, which also expressed stem cell antigen-1). Thus pdgfrα deletion differentially alters delta-like protein-1 and Sox9, suggesting that targeting different downstream pathways in PDGF-A-responsive LF could identify strategies that promote lung regeneration without initiating fibrosis.
Asunto(s)
Diferenciación Celular , Fibroblastos/citología , Lípidos/fisiología , Proteínas de Microfilamentos/fisiología , Proteínas Musculares/fisiología , Miofibroblastos/citología , Alveolos Pulmonares/citología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Animales , Biomarcadores/análisis , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Integrasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Miofibroblastos/metabolismo , Fenotipo , Alveolos Pulmonares/metabolismo , Mecánica Respiratoria , Factor de Transcripción SOX9/metabolismo , Transducción de SeñalRESUMEN
Alveolar septal formation is required to support the respiration of growing mammals; in humans effacement of the alveolar surface and impaired gas exchange are critical features of emphysema and pulmonary fibrosis. Platelet-derived growth factor-A (PDGF-A) and its receptor PDGF-receptor-α (PDGFRα) are required for secondary septal elongation in mice during postnatal days 4 through 12 and they regulate the proliferation and septal location of interstitial fibroblasts. We examined lung fibroblasts (LF) to learn whether PDGFRα expression distinguished a population of precursor cells, with enhanced proliferative and migratory capabilities. We identified a subpopulation of LF that expresses sonic hedgehog (Shh) and stem cell antigen-1 (Sca1). PDGF-A and Shh both increased cytokinesis and chemotaxis in vitro, but through different mechanisms. In primary LF cultures, Shh signaled exclusively through a noncanonical pathway involving generation of Rac1-GTP, whereas both the canonical and noncanonical pathways were used by the Mlg neonatal mouse LF cell line. LF preferentially oriented their primary cilia toward their anterior pole during migration. Furthermore, a larger proportion of PDGFRα-expressing LF, which are more abundant at the septal tips, bore primary cilia compared with other alveolar cells. In pulmonary emphysema, destroyed alveolar septa do not regenerate, in part because cells fail to assume a configuration that allows efficient gas exchange. Better understanding how LF are positioned during alveolar development could identify signaling pathways, which promote alveolar septal regeneration.
Asunto(s)
Proteínas Hedgehog/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Actinas/análisis , Animales , Antígenos CD34/análisis , Antígenos Ly/análisis , Línea Celular , Movimiento Celular , Proliferación Celular , Fibroblastos/metabolismo , Proteínas Hedgehog/análisis , Antígeno Ki-67/análisis , Pulmón/embriología , Pulmón/metabolismo , Proteínas de la Membrana/análisis , Ratones , Ratones Transgénicos , Neuropéptidos/biosíntesis , Neuropéptidos/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismo , Proteína de Unión al GTP rac1/biosíntesis , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BACKGROUND: Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation. METHODS: Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/G0 to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27(kip1) and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27(kip1) in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry. RESULTS: In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27(kip1) correlated with reduced LF entry into S-phase. CONCLUSIONS: Nuclear localization of FoxO3a, an important regulator of p27(kip1) gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Pulmón/citología , Pulmón/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Proteína Forkhead Box O3 , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Transgénicos , Puntos de Control de la Fase S del Ciclo Celular/fisiología , Transducción de Señal/fisiologíaRESUMEN
In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-Rα), is required for expansion, but the receptor's relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfrα-expressing LF (pdgfrα-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-Rα-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (αSMA) producing pdgfrα-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-Rα gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.
Asunto(s)
Miofibroblastos/enzimología , Alveolos Pulmonares/enzimología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Actinas/metabolismo , Factores de Edad , Envejecimiento , Análisis de Varianza , Animales , Animales Recién Nacidos , Apoptosis , Benzamidas , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Mesilato de Imatinib , Ratones , Ratones Transgénicos , Miofibroblastos/patología , FN-kappa B/metabolismo , Fosforilación , Piperazinas/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/patología , Pirimidinas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Transducción de Señal , Activación TranscripcionalRESUMEN
A feline immunodeficiency virus (FIV)-based lentiviral vector was pseudotyped to identify envelope (env) glycoproteins that direct efficient gene transfer to pulmonary epithelia for the treatment or prevention of lung diseases. The envelope glycoprotein from the Jaagsiekte sheep retrovirus (JSRV) is a candidate under investigation. We utilized high titer FIV vector (>10(8) TU/ml) pseudotyped with the JSRV env glycoprotein (JSRVFIV) to study the transduction of polarized primary cultures of human airway epithelia and receptor/vector interactions. The reported receptor for JSRV, hyaluronidase 2 (HYAL2), is a GPI-linked protein. We expressed FLAG-tagged HYAL2 in polarized airway epithelia using an adenoviral vector and documented that the HYAL2 protein sorts predominantly to the apical surface. Of interest, the efficiency of gene transfer with apically applied JSRV-FIV was markedly less than FIV pseudotyped with VSV-G, even in Ad-HYAL2 complemented epithelia. The inefficient gene transfer with JSRV-FIV in HYAL2 complemented cells suggests that factors other than receptor abundance limit apical gene transfer efficiency with this envelope. JSRV-FIV transduced the distal lung epithelia of rabbits in vivo and transduced primary cultures of rabbit type II cells with 100-fold greater efficiency than primary cultures of rabbit tracheal cells. These data indicate that a lentivirus pseudotyped with the JSRV envelope glycoprotein transduces type II cells with greater efficiency than conducting airway epithelia and provides an example of glycoprotein-mediated cell-specific tropism within a tissue with a widely heterogeneous cell population.
Asunto(s)
Retrovirus Ovino Jaagsiekte/genética , Lentivirus/genética , Mucosa Respiratoria/fisiología , Transducción Genética/métodos , Proteínas del Envoltorio Viral/genética , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Transformada , Células Cultivadas , Proteínas Ligadas a GPI , Vectores Genéticos/genética , Glicoproteínas/genética , Humanos , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Conejos , Mucosa Respiratoria/virología , Ovinos , Tráquea/citología , Tráquea/virología , TropismoRESUMEN
Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.
Asunto(s)
Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Esfingolípidos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Fenómenos Biofísicos , Biofisica , Permeabilidad Capilar/efectos de los fármacos , Ceramidas/farmacología , Colorantes/farmacocinética , Azul de Evans/farmacocinética , Masculino , Circulación Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Esfingomielina Fosfodiesterasa/metabolismo , Tensión SuperficialRESUMEN
Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Cardiolipinas/fisiología , Lesión Pulmonar/etiología , Neumonía Bacteriana/complicaciones , Animales , Sitios de Unión , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Pulmón/metabolismo , Pulmón/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Transferencia de Fosfolípidos , Neumonía Bacteriana/metabolismo , Surfactantes Pulmonares/metabolismoRESUMEN
Surfactant, highly enriched with phosphatidylcholine (PC), is secreted into the airspace by a classic apical secretory route, thereby maintaining lung stability. Herein, we show that adenoviral infection decreases surfactant PC in lungs by inhibiting its apical secretion and redirecting its export in alveolar cells by a basolateral route. These effects were not observed with replication-deficient adenovirus (Ad), specifically lacking early region 1 (E1) gene products. Adenoviral stimulation of basolateral PC export from cells was not observed after pharmacologic inhibition of ATP-binding cassette proteins, after introduction of small interfering RNA to the lipid pump ATP-binding cassette transporter A1 (ABCA1) or in ABCA1-defective human Tangier disease fibroblasts. Adenovirus and its E1A gene product increased ABCA1 levels by transcriptionally activating the ABCA1 gene. Thus, Ad lowers surfactant, in part, by triggering ABCA1-directed basolateral PC export, thereby limiting the cellular pool of surfactant PC destined for apical secretion. The results support a novel pathway, whereby a viral pathogen disrupts surfactant trafficking.
Asunto(s)
Adenoviridae/metabolismo , Fosfolípidos/química , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Tensoactivos/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Lung development is associated with a surge in surfactant phosphatidylcholine (PC) production to prepare the newborn for extrauterine breathing. This process is associated with a marked increase in the activity of the rate-regulatory surfactant enzyme, CTP:phosphocholine cytidylyltransferase (CCTalpha). To investigate the molecular basis for developmental activation of CCTalpha, we analyzed expression of endogenous CCTalpha and a reporter gene, beta-galactosidase, in fetal, newborn, and adult promoter-reporter transgenic mice. Transgenics harboring approximately 2 kb of the CCTalpha promoter linked upstream of a beta-galactosidase reporter gene displayed relatively high expression in distal lung epithelia. Endogenous lung CCTalpha and beta-galactosidase activities, protein content, and transcript levels displayed maximal expression within the newborn period. CCTalpha and beta-galactosidase activities and enzyme levels increased with time in cultured fetal lung explants isolated from transgenics. Transfectional analysis using CCTalpha promoter-reporter constructs in developing rat type II cells revealed that a region encompassing -169/+71 contained the DNA elements required for perinatal activation. The studies demonstrate that developmental induction of surfactant phospholipid is due, at least in part, to transcriptional activation of the CCTalpha gene.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Pulmón/crecimiento & desarrollo , Animales , Células Cultivadas , Genes Reporteros , Pulmón/citología , Pulmón/enzimología , Ratones , Ratones Transgénicos , Fosfolípidos/metabolismo , Regiones Promotoras Genéticas , Alveolos Pulmonares/citología , Alveolos Pulmonares/enzimología , Surfactantes Pulmonares/metabolismo , Ratas , Transcripción Genética , Transfección , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.
Asunto(s)
Células Epiteliales/citología , Pulmón/patología , Oxidorreductasas/química , Esfingolípidos/metabolismo , Animales , Western Blotting , Membrana Celular/metabolismo , Ceramidas/metabolismo , Clonación Molecular , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Vectores Genéticos , Hidrólisis , Pulmón/citología , Pulmón/embriología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Fosfatidilcolinas/metabolismo , Plásmidos/metabolismo , Isoformas de Proteínas , ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Factores de Tiempo , TransfecciónRESUMEN
Tumor necrosis factor (TNF)-alpha is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-alpha decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-alpha, we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-alpha overexpression. Compared with controls, TNF-alpha transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-alpha results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.
Asunto(s)
Colina/análogos & derivados , Pulmón/metabolismo , Fosfatidilcolinas/farmacocinética , Surfactantes Pulmonares/farmacocinética , Factor de Necrosis Tumoral alfa/genética , Animales , Western Blotting , Colina/farmacocinética , Colina Quinasa/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Ácidos Grasos/metabolismo , Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfolípidos/metabolismo , ARN Mensajero/análisis , Esfingolípidos/metabolismo , TritioRESUMEN
We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.