Alexis N. Brumwell

Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix

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 β-galactosidase (β-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-β1. β-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-β1 expression. The increase in vimentin-positive cells within injured lungs was nearly all β-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-β1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-β1 pathway and, if exposed to active TGF-β1, 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.

Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis

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.

Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice

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 α6β4, 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 β4 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 β4+ 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.

Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury

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.

Integrin α3β1–dependent β-catenin phosphorylation links epithelial Smad signaling to cell contacts

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, α3β1, were found to have a markedly blunted EMT response to TGF-β1. A mechanism for this defect was explored in α3-null cells reconstituted with wild-type (wt) α3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-β1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-β1 receptors, generate phospho-Smad2 (p-Smad2)–pY654–β-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2–pY654–β-catenin complexes do not form in the absence of α3 or when α3β1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that α3β1 coordinates cross talk between β-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-β1 activation.

Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with β1 integrins

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 β1 integrin interactions but maintain uPA binding, vitronectin attachment and association with αV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with β1 integrins recapitulated previously reported findings with β1-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–αVβ5-integrin, rather than through the uPAR–α3β1-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–β1-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.

Inhibition of Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis by Methacycline

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)-β1-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-β1-induced α-smooth muscle actin, Snail1, and collagen I of primary alveolar epithelial cells . Methacycline inhibited TGF-β1-induced non-Smad pathways, including c-Jun N-terminal kinase, p38, and Akt activation, but not Smad or β-catenin transcriptional activity. Methacycline had no effect on baseline c-Jun N-terminal kinase, p38, or Akt activities or lung fibroblast responses to TGF-β1. 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-β1-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.

Identification of pY654-β-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia

Hypoxia is linked to epithelial–mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of β-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-β-catenin and HIF1α. All pY654-β-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for β-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-β-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated β-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-β-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-β-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung

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 that promotes apoptosis if ER stress remains high (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 importance of IRE1α activation in 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.

Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype

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.