Lef-1 controls cell cycle progression in airway basal cells to regulate proliferation and differentiation.

The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine.

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

Depletion of Airway Submucosal Glands and TP63+KRT5+ Basal Cells in Obliterative Bronchiolitis.

RATIONALE: Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB. OBJECTIVES: Two major SC compartments in airways are submucosal glands (SMGs) and surface airway p63 (also known as TP63 [tumor protein 63])-positive/K5 (also known as KRT5 [keratin 5])-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB. METHODS: Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay. MEASUREMENTS AND MAIN RESULTS: Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63+/K5+ and increase in K5+/K14+ and K14+ BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model. CONCLUSIONS: SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.

Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells.

Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFß/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs.

Mucociliary clearance (MCC) and submucosal glands are major components of airway innate immunity that have impaired function in cystic fibrosis (CF). Although both of these defense systems develop postnatally in the ferret, the lungs of newborn ferrets remain sterile in the presence of a functioning cystic fibrosis transmembrane conductance regulator gene. We evaluated several components of airway innate immunity and inflammation in the early CF ferret lung. At birth, the rates of MCC did not differ between CF and non-CF animals, but the height of the airway surface liquid was significantly reduced in CF newborn ferrets. CF ferrets had impaired MCC after 7 days of age, despite normal rates of ciliogenesis. Only non-CF ferrets eradicated Pseudomonas directly introduced into the lung after birth, whereas both genotypes could eradicate Staphylococcus. CF bronchoalveolar lavage fluid (BALF) had significantly lower antimicrobial activity selectively against Pseudomonas than non-CF BALF, which was insensitive to changes in pH and bicarbonate. Liquid chromatography-tandem mass spectrometry and cytokine analysis of BALF from sterile Caesarean-sectioned and nonsterile naturally born animals demonstrated CF-associated disturbances in IL-8, TNF-α, and IL-ß, and pathways that control immunity and inflammation, including the complement system, macrophage functions, mammalian target of rapamycin signaling, and eukaryotic initiation factor 2 signaling. Interestingly, during the birth transition, IL-8 was selectively induced in CF BALF, despite no genotypic difference in bacterial load shortly after birth. These results suggest that newborn CF ferrets have defects in both innate immunity and inflammatory signaling that may be important in the early onset and progression of lung disease in these animals.

CcpA-independent glucose regulation of lactate dehydrogenase 1 in Staphylococcus aureus.

Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (P(ldh1)). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for P(ldh1). A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in P(ldh1). However, CcpA had no affinity for P(ldh1) in vitro and a genomic mutation of CRE upstream of P(ldh1) in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at P(ldh1).