Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury

Ievlev, Vitaly; Jensen-Cody, Chandler Walker; Lynch, Thomas J.; Pai, Albert; Park, Soo Hyung; Shahin, Weam; Wang, Kai; Parekh, Kalpaj R.; Engelhardt, John F.

doi:10.1093/stmcls/sxac038

Cited by 7 publications

(9 citation statements)

References 51 publications

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“…Thus, we conclude that glandular contribution to the airway surface repair can at least partially account for appearance of Krt14 on the injured surface epithelium. These observations are consistent with prior work demonstrating surface repair by glandular progenitors in mice and ferrets in vivo ( 8 , 9 , 24 , 25 ).…”

Section: Resultssupporting

confidence: 92%

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

Ievlev¹,

Lynch²,

Freischlag³

et al. 2023

JCI Insight

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Keratin expression dynamically changes in airway basal cells (BCs) following acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In Bronchiolitis Obliterans (BO) following lung transplantation, BC clonogenicity declines which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated the roles of these keratins using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs failed to differentiate into club and ciliated cells, but had enhanced clonogenicity.By contrast, Krt15-KO did not alter BC differentiation, but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo following injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, while dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts that of Krt14-KO and resembles the phenotype in BO with decreased clonogenicity, increased Krt14 and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior which is relevant in chronic disease states like BO.

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Section: Resultssupporting

confidence: 92%

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

Ievlev¹,

Lynch²,

Freischlag³

et al. 2023

JCI Insight

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“…However, in the silica-treated group, basal cells were distributed not only on the basal layer but also on the upper layer, but their ectopic distribution was restored by treatment with the NLRP3 inflammasome inhibitor MCC950 (Figure 6 A). Furthermore, SOX2 and SOX9 are two pluripotency-associated stem/progenitor cell transcription factors that drive the proliferation and differentiation of airway epithelial basal cells following injury 51 - 53 . Our immunofluorescence results indicated that after stimulation with silica, most ectopic basal cells on the upper layer were positive for SOX2 (Figure 6 B, white arrows), with a significantly increased proportion of SOX2 + basal cells in the LSPC-derived airway epithelium (Figure 6 C).…”

Section: Resultsmentioning

confidence: 99%

“…The specific spatiotemporal distribution of SOX9 and SOX2 contributes to proper cell proliferation and differentiation, allowing lung development, repair and regeneration. In response to injury caused by smoking, SOX9 drives the generation of airway basal cells and leads to the repair of the mucociliary epithelium 52 , 53 . In bleomycin-induced lung injury, SOX2 regulates alveolar epithelial plasticity by inducing the emergence of progenitor-like cells, but continuous proliferation of SOX2 + cells disrupts alveolar structure, including septal rupture 90 .…”

Section: Discussionmentioning

confidence: 99%

NLRP3 Inflammasome Mediates Silica-induced Lung Epithelial Injury and Aberrant Regeneration in Lung Stem/Progenitor Cell-derived Organotypic Models

Zhao¹,

Zhang²,

Wen³

et al. 2023

Int. J. Biol. Sci.

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Silica-induced lung epithelial injury and fibrosis are vital pathogeneses of silicosis. Although the NOD-like receptor protein 3 (NLRP3) inflammasome contributes to silica-induced chronic lung inflammation, its role in epithelial injury and regeneration remains unclear. Here, using mouse lung stem/progenitor cell-derived organotypic systems, including 2D air-liquid interface and 3D organoid cultures, we investigated the effects of the NLRP3 inflammasome on airway epithelial phenotype and function, cellular injury and regeneration, and the potential mechanisms. Our data showed that silica-induced NLRP3 inflammasome activation disrupted the epithelial architecture, impaired mucociliary clearance, induced cellular hyperplasia and the epithelial-mesenchymal transition in 2D culture, and inhibited organoid development in 3D system. Moreover, abnormal expression of the stem/progenitor cell markers SOX2 and SOX9 was observed in the 2D and 3D organotypic models after sustained silica stimulation. Notably, these silica-induced structural and functional abnormalities were ameliorated by MCC950, a selective NLRP3 inflammasome inhibitor. Further studies indicated that the NF-κB, Shh-Gli and Wnt/β-catenin pathways were involved in NLRP3 inflammasome-mediated abnormal differentiation and dysfunction of the airway epithelium. Thus, prolonged NLRP3 inflammasome activation caused injury and aberrant lung epithelial regeneration, suggesting that the NLRP3 inflammasome is a pivotal target for regulating tissue repair in chronic inflammatory lung diseases.

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“…DMEM was chosen for its simplicity because we hypothesized that tracheal explants can produce everything that they require for prolonged survival and repair after injury. F-medium was chosen because it is permissive for the survival of both epithelial and mesenchymal cells when used in colony formation efficiency (CFE) assays of airway basal cells that are grown on 3 T3-J2 fibroblasts ( 15 , 16 ).…”

Section: Resultsmentioning

confidence: 99%

“…Two of them were MMP9 and LAMA3 that are both associated with ECM deposition and remodeling and are upregulated in epidermal wound healing ( 25 , 26 ). We also assessed the expression of SOX9, a marker of airway SMG progenitors that have been previously shown to participate in SAE regeneration ( 15 , 27 , 28 ). As expected, all three of these targets were upregulated in both severely injured and moderately injured epithelia, with the level of upregulation corresponding to the level of injury ( Figure 6D ).…”

Section: Resultsmentioning

confidence: 99%

Development and characterization of ferret ex vivo tracheal injury and cell engraftment model

et al. 2023

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The field of airway biology research relies primarily on in vitro and in vivo models of disease and injury. The use of ex vivo models to study airway injury and cell-based therapies remains largely unexplored although such models have the potential to overcome certain limitations of working with live animals and may more closely replicate in vivo processes than in vitro models can. Here, we characterized a ferret ex vivo tracheal injury and cell engraftment model. We describe a protocol for whole-mount staining of cleared tracheal explants, and showed that it provides a more comprehensive structural overview of the surface airway epithelium (SAE) and submucosal glands (SMGs) than 2D sections, revealing previously underappreciated structural anatomy of tracheal innervation and vascularization. Using an ex vivo model of tracheal injury, we evaluated the injury responses in the SAE and SMGs that turned out to be consistent with published in vivo work. We used this model to assess factors that influence engraftment of transgenic cells, providing a system for optimizing cell-based therapies. Finally, we developed a novel 3D-printed reusable culture chamber that enables live imaging of tracheal explants and differentiation of engrafted cells at an air-liquid interface. These approaches promise to be useful for modeling pulmonary diseases and testing therapies.Graphical abstract1,2. We describe here a method for differential mechanical injury of ferret tracheal explants that can be used to evaluate airway injury responses ex vivo. 3. Injured explants can be cultured at ALI (using the novel tissue-transwell device on the right) and submerged long-term to evaluate tissue-autonomous regeneration responses. 4. Tracheal explants can also be used for low throughput screens of compounds to improve cell engraftment efficiency or can be seeded with particular cells to model a disease phenotype. 5. Lastly, we demonstrate that ex vivo-cultured tracheal explants can be evaluated by various molecular assays and by immunofluorescent imaging that can be performed live using our custom-designed tissue-transwell.

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Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury

Cited by 7 publications

References 51 publications

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

NLRP3 Inflammasome Mediates Silica-induced Lung Epithelial Injury and Aberrant Regeneration in Lung Stem/Progenitor Cell-derived Organotypic Models

Development and characterization of ferret ex vivo tracheal injury and cell engraftment model

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