RESUMEN
The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.
Asunto(s)
Bronquiolos , Hurones , Células Madre Multipotentes , Alveolos Pulmonares , Animales , Bronquiolos/citología , Linaje de la Célula , Humanos , Pulmón/patología , Ratones , Células Madre Multipotentes/citología , Alveolos Pulmonares/citología , Enfermedad Pulmonar Obstructiva CrónicaRESUMEN
BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.
Asunto(s)
Compuestos de Anilina , Fibrosis Pulmonar , Sulfonamidas , Ratones , Animales , Fibrosis Pulmonar/metabolismo , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Estrés del Retículo Endoplásmico , Ratones Noqueados , Colágeno/metabolismo , Bleomicina/farmacologíaRESUMEN
We recently identified epigallocatechin gallate (EGCG), a trihydroxyphenolic compound, as a dual inhibitor of lysyl oxidase-like2 and transforming growth factor-ß1 (TGFß1) receptor kinase that when given orally to patients with idiopathic pulmonary fibrosis (IPF) reversed profibrotic biomarkers in their diagnostic biopsies. Here, we extend these findings to advanced pulmonary fibrosis using cultured precision-cut lung slices from explants of patients with IPF undergoing transplantation. During these experiments, we were surprised to discover that not only did EGCG attenuate TGFß1 signalling and new collagen accumulation but also activated matrix metalloproteinase-dependent collagen I turnover, raising the possibility of slow fibrosis resolution with continued treatment.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Colágeno Tipo I/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Humanos , Fibrosis Pulmonar Idiopática/patología , Immunoblotting , Pulmón/patología , Transducción de SeñalRESUMEN
Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/patología , Lesión Pulmonar/patología , Pulmón/citología , Pulmón/patología , Repitelización , Células Madre/citología , Animales , Bleomicina , Linaje de la Célula , Proliferación Celular , Separación Celular , Quistes/metabolismo , Quistes/patología , Células Epiteliales/metabolismo , Femenino , Humanos , Queratina-5/metabolismo , Pulmón/fisiología , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/virología , Masculino , Ratones , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Trasplante de Células Madre , Células Madre/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
A high-throughput small-molecule screen was conducted to identify inhibitors of epithelial-mesenchymal transition (EMT) that could be used as tool compounds to test the importance of EMT signaling in vivo during fibrogenesis. Transforming growth factor (TGF)-ß1-induced fibronectin expression and E-cadherin repression in A549 cells were used as 48-hour endpoints in a cell-based imaging screen. Compounds that directly blocked Smad2/3 phosphorylation were excluded. From 2,100 bioactive compounds, methacycline was identified as an inhibitor of A549 EMT with the half maximal inhibitory concentration (IC50) of roughly 5 µM. In vitro, methacycline inhibited TGF-ß1-induced α-smooth muscle actin, Snail1, and collagen I of primary alveolar epithelial cells . Methacycline inhibited TGF-ß1-induced non-Smad pathways, including c-Jun N-terminal kinase, p38, and Akt activation, but not Smad or ß-catenin transcriptional activity. Methacycline had no effect on baseline c-Jun N-terminal kinase, p38, or Akt activities or lung fibroblast responses to TGF-ß1. In vivo, 100 mg/kg intraperitoneal methacycline delivered daily beginning 10 days after intratracheal bleomycin improved survival at Day 17 (P < 0.01). Bleomycin-induced canonical EMT markers, Snail1, Twist1, collagen I, as well as fibronectin protein and mRNA, were attenuated by methacycline (Day 17). Methacycline did not attenuate inflammatory cell accumulation or alter TGF-ß1-responsive genes in alveolar macrophages. These studies identify a novel inhibitor of EMT as a potent suppressor of fibrogenesis, further supporting the concept that EMT signaling is important to lung fibrosis. The findings also provide support for testing the impact of methacycline or doxycycline, an active analog, on progression of human pulmonary fibrosis.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Metaciclina/farmacología , Alveolos Pulmonares/efectos de los fármacos , Fibrosis Pulmonar/tratamiento farmacológico , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular , Colágeno Tipo I/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/metabolismo , Fibrosis Pulmonar/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFß1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.
RESUMEN
Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate this, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Unexposed biopsy samples showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG significantly downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted Frizzle-like Receptor Protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s). In human AEC2-fibroblast coculture organoids, sFRP2 was essential for AEC2 trans-differentiation to basal cells. Precision cut lung slices (PCLS) from normal donors demonstrated that TGFß1 promoted KRT17 expression and AEC2 morphological change, while sFRP2 was necessary for KRT5 expression in AEC2-derived basaloid cells. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin-related signaling in AEC2s were required for sFRP2-induced KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify the TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.
RESUMEN
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-beta1. As the ECM critically regulates AEC responses to TGF-beta1, we explored the role of the prominent epithelial integrin alpha3beta1 in experimental fibrosis by generating mice with lung epithelial cell-specific loss of alpha3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through beta-catenin has been implicated in EMT; we found that in primary AECs, alpha3 integrin was required for beta-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-beta-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-beta-catenin/pSmad2 complexes and showed accumulation of pY654-beta-catenin in myofibroblasts. These findings demonstrate epithelial integrin-dependent profibrotic crosstalk between beta-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.
Asunto(s)
Células Epiteliales/fisiología , Fibroblastos/metabolismo , Integrina alfa3beta1/metabolismo , Fibrosis Pulmonar , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , beta Catenina/metabolismo , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Células Cultivadas , Células Epiteliales/citología , Fibroblastos/citología , Humanos , Integrina alfa3beta1/genética , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Mesodermo/citología , Mesodermo/fisiología , Ratones , Ratones Transgénicos , Fenotipo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteína Smad2/genéticaRESUMEN
Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-ß1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.
Asunto(s)
Transdiferenciación Celular/fisiología , Células Epiteliales/citología , Fibrosis Pulmonar Idiopática/patología , Queratina-5/metabolismo , Alveolos Pulmonares/citología , Mucosa Respiratoria/citología , Células Epiteliales Alveolares/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Células Cultivadas , Células Epidérmicas/citología , Fibroblastos/citología , Humanos , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Transducción de Señal/fisiología , Análisis de la Célula Individual , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Lung injury activates specialized adult epithelial progenitors to regenerate the epithelium. Depending on the extent of injury, both remaining alveolar type II cells (AEC2s) and distal airway stem/progenitors mobilize to cover denuded alveoli and restore normal barriers. The major source of airway stem/progenitors other than basal-like cells remains uncertain. Here, we define a distinct subpopulation (â¼5%) of club-like lineage-negative epithelial progenitors (LNEPs) marked by high H2-K1 expression critical for alveolar repair. Quiescent H2-K1high cells account for virtually all in vitro regenerative activity of airway lineages. After bleomycin injury, H2-K1 cells expand and differentiate in vivo to alveolar lineages. However, injured H2-K1 cells eventually develop impaired self-renewal with features of senescence, limiting complete repair. Normal H2-K1high cells transplanted into injured lungs differentiate into alveolar cells and rescue lung function. These findings indicate that small subpopulations of specialized stem/progenitors are required for effective lung regeneration and are a potential therapeutic adjunct after major lung injury.
Asunto(s)
Células Epiteliales , Lesión Pulmonar , Células Epiteliales Alveolares , Diferenciación Celular , Humanos , Pulmón , Células MadreRESUMEN
Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.
Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Endorribonucleasas/antagonistas & inhibidores , Fibrosis/tratamiento farmacológico , Pulmón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibrosis/metabolismo , Fibrosis/patología , Pulmón/metabolismo , Pulmón/patología , Ratones , Inhibidores de Proteínas Quinasas/uso terapéutico , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+ cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.
Asunto(s)
Senescencia Celular , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/patología , Leucotrienos/metabolismo , Pulmón/patología , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Bleomicina/toxicidad , Líquido del Lavado Bronquioalveolar/química , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/diagnóstico , Leucotrienos/análisis , Inhibidores de la Lipooxigenasa/farmacología , Pulmón/citología , Masculino , Ratones , Cultivo Primario de Células , Prostaglandinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.
Asunto(s)
Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Alveolos Pulmonares/metabolismo , Enfisema Pulmonar/metabolismo , Transducción de Señal , Animales , Fibroblastos/patología , Proteínas Hedgehog/genética , Humanos , Ratones , Ratones Noqueados , Alveolos Pulmonares/patología , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologíaRESUMEN
After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/ß-catenin activity in Sox2pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.
Asunto(s)
Linaje de la Célula , Proliferación Celular , Transdiferenciación Celular , Células Epiteliales/metabolismo , Hipoxia/metabolismo , Gripe Humana/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Oxígeno/metabolismo , Alveolos Pulmonares/metabolismo , Regeneración , Animales , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Hipoxia/genética , Hipoxia/patología , Hipoxia/virología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/patología , Gripe Humana/virología , Queratina-5/genética , Queratina-5/metabolismo , Masculino , Ratones Transgénicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , Receptores Notch/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula Individual , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Vía de Señalización WntRESUMEN
Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6ß4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin ß4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified ß4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.
Asunto(s)
Células Epiteliales/fisiología , Integrina alfa6beta4/metabolismo , Pulmón/anatomía & histología , Pulmón/fisiología , Regeneración/fisiología , Mucosa Respiratoria/citología , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/citología , Femenino , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Células Madre/citología , Células Madre/fisiología , Uteroglobina/metabolismoRESUMEN
Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, alpha3beta1, were found to have a markedly blunted EMT response to TGF-beta1. A mechanism for this defect was explored in alpha3-null cells reconstituted with wild-type (wt) alpha3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-beta1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-beta1 receptors, generate phospho-Smad2 (p-Smad2)-pY654-beta-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2-pY654-beta-catenin complexes do not form in the absence of alpha3 or when alpha3beta1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that alpha3beta1 coordinates cross talk between beta-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-beta1 activation.
Asunto(s)
Integrina alfa3beta1/metabolismo , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , beta Catenina/metabolismo , Animales , Adhesión Celular , Células Epiteliales/metabolismo , Epitelio/metabolismo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Transgénicos , Fosforilación , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate beta1 integrin interactions but maintain uPA binding, vitronectin attachment and association with alphaV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with beta1 integrins recapitulated previously reported findings with beta1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR-alphaVbeta5-integrin, rather than through the uPAR-alpha3beta1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR-beta1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo.
Asunto(s)
Integrina beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Integrina beta1/genética , Neoplasias Pulmonares/genética , Ratones , Ratones Desnudos , Mutación Puntual , Unión Proteica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing beta-galactosidase (beta-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-beta1. beta-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-beta1 expression. The increase in vimentin-positive cells within injured lungs was nearly all beta-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-beta1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-beta1 pathway and, if exposed to active TGF-beta1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.