BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

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.

Human distal airways contain a multipotent secretory cell that can regenerate alveoli.

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.

Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells.

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.

Alveolar regeneration through a Krt8+ transitional stem cell state that persists in human lung fibrosis.

The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues.

VEGF Drives the Car toward Better Gas Exchange.

How lung epithelium and endothelium co-develop to maintain structural integrity of alveoli remains unclear. In this issue of Developmental Cell, Ellis et al. define how epithelial Vegfa directs development of a distinct endothelial cell population that ultimately plays a critical role in ensuring appropriate alveolar septation during alveologenesis.

Distinct Airway Epithelial Stem Cells Hide among Club Cells but Mobilize to Promote Alveolar Regeneration.

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 (∼5%) 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.

PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer.

PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. Abbreviations AKT V-Akt Murine Thymoma Viral Oncogene CA Cancer adjacent CDK1 Cyclin dependent kinase 1 CENPC-C Centromere Protein C ChIP Chromatin Immunoprecipitation co-IP Co-immunoprecipitation COSMIC Catalog of Somatic Mutations In Cancer CREB cAMP Responsive Element Binding Protein C-tail Carboxy terminal tail E2F1 E2F Transcription Factor 1 ECIS Electric Cell-substrate Impedance Sensing EGFR Epidermal Growth Factor Receptor GSI Gamma Secretase Inhibitor HDAC1 Histone Deacetylase 1 HP1 Heterochromatin protein 1 KAP1/TRIM28 KRAB-Associated Protein 1/Tripartite Motif Containing 28 MAF1 Repressor of RNA polymerase III transcription MAF1 homolog MCM2 Minichromosome Maintenance Complex Component 2 miRNA micro RNA MTF1 Metal-Regulatory Transcription Factor 1 PARP Poly(ADP-Ribose) Polymerase PD-1 Programmed Cell Death 1 PD-L1 Programmed Cell Death 1 Ligand 1 PI3K Phosphatidylinositol-4,5-Bisphosphate 3-Kinase PLK Polo-like Kinase pPTEN Phosphorylated PTEN PTEN Phosphatase and Tensin Homolog deleted on chromosome ten PTM Post Translational Modification Rad51 RAD51 Recombinase Rad52 RAD52 Recombinase RPA1 Replication protein A SILAC Stable Isotope Labeling with Amino Acids in Cell Culture SRF Serum Response Factor TKI Tyrosine Kinase inhbitors TMA Tissue Microarray TOP2A DNA Topoisomerase 2A.

Galectin-1 inhibition attenuates profibrotic signaling in hypoxia-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by lung remodeling arising from epithelial injury, aberrant fibroblast growth, and excessive deposition of extracellular matrix. Repeated epithelial injury elicits abnormal wound repair and lung remodeling, often associated with alveolar collapse and edema, leading to focal hypoxia. Here, we demonstrate that hypoxia is a physiological insult that contributes to pulmonary fibrosis (PF) and define its molecular roles in profibrotic activation of lung epithelial cells. Hypoxia increased transcription of profibrotic genes and altered the proteomic signatures of lung epithelial cells. Network analysis of the hypoxic epithelial proteome revealed a crosstalk between transforming growth factor-ß1 and FAK1 (focal adhesion kinase-1) signaling, which regulated transcription of galectin-1, a profibrotic molecule. Galectin-1 physically interacted with and activated FAK1 in lung epithelial cells. We developed a novel model of exacerbated PF wherein hypoxia, as a secondary insult, caused PF in mice injured with subclinical levels of bleomycin. Hypoxia elevated expression of phosphorylated FAK1, galectin-1, and α-smooth muscle actin and reduced caspase-3 activation, suggesting aberrant injury repair. Galectin-1 inhibition caused apoptosis in the lung parenchyma and reduced FAK1 activation, preventing the development of hypoxia-induced PF. Galectin-1 inhibition also attenuated fibrosis-associated lung function decline. Further, galectin-1 transcript levels were increased in the lungs of IPF patients. In summary, we have identified a profibrotic role of galectin-1 in hypoxia signaling driving PF.

Data on evolution of intrinsically disordered regions of the human kinome and contribution of FAK1 IDRs to cytoskeletal remodeling.

We present data on the evolution of intrinsically disordered regions (IDRs) taking into account the entire human protein kinome. The evolutionary data of the IDRs with respect to the kinase domains (KDs) and kinases as a whole protein (WP) are reported. Further, we have reported its post translational modifications of FAK1 IDRs and their contribution to the cytoskeletal remodeling. We also report the data to build a protein-protein interaction (PPI) network of primary and secondary FAK1-interacting hybrid proteins. Detailed analysis of the data and its effect on FAK1-related functions have been described in "Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling" (Kathiriya et. al., 2016) [1].

Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling.

Therapeutic protein kinase inhibitors are designed on the basis of kinase structures. Here, we define intrinsically disordered regions (IDRs) in structurally hybrid kinases. We reveal that 65% of kinases have an IDR adjacent to their kinase domain (KD). These IDRs are evolutionarily more conserved than IDRs distant to KDs. Strikingly, 36 kinases have adjacent IDRs extending into their KDs, defining a unique structural and functional subset of the kinome. Functional network analysis of this subset of the kinome uncovered FAK1 as topologically the most connected hub kinase. We identify that KD-flanking IDR of FAK1 is more conserved and undergoes more post-translational modifications than other IDRs. It preferentially interacts with proteins regulating scaffolding and kinase activity, which contribute to cytoskeletal remodeling. In summary, spatially and evolutionarily conserved IDRs in kinases may influence their functions, which can be exploited for targeted therapies in diseases including those that involve aberrant cytoskeletal remodeling.

Presence and utility of intrinsically disordered regions in kinases.

Since aberrant cell signaling pathways underlie majority of pathophysiological morbidities, kinase inhibitors are routinely used for pharmacotherapy. However, most kinase inhibitors suffer from adverse off-target effects. Inhibition of one kinase in a pathogenic signaling pathway elicits multiple compensatory feedback signaling loops, reinforcing the pathway rather than inhibiting it, leading to chemoresistance. Thus, development of novel computational strategies providing predictive evidence to inhibit a specific set of kinases to mitigate an aberrant signaling pathway with minimum side-effects is imperative. First, our analyses reveal that many kinases contain intrinsically disordered regions, which may participate in facilitating protein-protein interactions at the kinome level. Second, we employ a kinome-wide approach to identify intrinsic disorder and streamline a methodology that adds to the knowledge of therapeutically targeting kinase cascades to treat diseases. Furthermore, we find that within the kinome network, some kinases with intrinsically disordered regions have a high topological score, likely acting as kinome modulators. Third, using network analysis, we demonstrate that 5 kinases emerge as topologically most significant, forming kinome sub-networks, comprising of other kinases and transcription factors that are known to serve as drivers of disease pathogenesis. To support these findings, we have biologically validated the interplay between kinome modulators SRC and AKT kinases and uncovered their novel function in regulating transcription factors of the SMAD family. Taken together, we identify novel kinome modulators driven by intrinsic disorder, and biologically validate the thesis that therapeutic disruption of the function of kinome modulators engaged in regulatory cross-talk between disparate pathways can lead to reduced oncogenic potential in cancer cells.