Pulmonary neuroendocrine cells: physiology, tissue homeostasis and disease.

Mammalian lungs have the ability to recognize external environments by sensing different compounds in inhaled air. Pulmonary neuroendocrine cells (PNECs) are rare, multi-functional epithelial cells currently garnering attention as intrapulmonary sensors; PNECs can detect hypoxic conditions through chemoreception. Because PNEC overactivation has been reported in patients suffering from respiratory diseases - such as asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia and other congenital diseases - an improved understanding of the fundamental characteristics of PNECs is becoming crucial in pulmonary biology and pathology. During the past decade, murine genetics and disease models revealed the involvement of PNECs in lung ventilation dynamics, mechanosensing and the type 2 immune responses. Single-cell RNA sequencing further unveiled heterogeneous gene expression profiles in the PNEC population and revealed that a small number of PNECs undergo reprogramming during regeneration. Aberrant large clusters of PNECs have been observed in neuroendocrine tumors, including small-cell lung cancer (SCLC). Modern innovation of imaging analyses has enabled the discovery of dynamic migratory behaviors of PNECs during airway development, perhaps relating to SCLC malignancy. This Review summarizes the findings from research on PNECs, along with novel knowledge about their function. In addition, it thoroughly addresses the relevant questions concerning the molecular pathology of pulmonary diseases and related therapeutic approaches.

Phosphorylation and dephosphorylation of Ser852 and Ser889 control the clustering, localization and function of PAR3.

Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.

Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells.

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of ß-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

The Epithelial Circumferential Actin Belt Regulates YAP/TAZ through Nucleocytoplasmic Shuttling of Merlin.

Circumferential actin belts underlying the adherens junctions of columnar epithelial cell monolayers control intercellular surface tension and cell shape to maintain tissue integrity. Yes-associated protein (YAP) and its paralog TAZ are proliferation-activating transcriptional coactivators that shuttle between the nucleus and cytoplasm. Previous studies suggest the importance of stress fibers in the actin cytoskeleton for regulation of YAP nuclear localization; however, the role of the circumferential actin belt on YAP localization remains unclarified. By manipulating actin tension, we demonstrate that circumferential actin belt tension suppresses YAP/TAZ nuclear localization. This suppression requires Merlin, an F-actin binding protein associated with adherens junctions. Merlin physically interacts with YAP/TAZ, and nuclear export sequences of Merlin are required for suppression. Together, with the observation that the association between E-cadherin and Merlin was diminished by tension in circumferential actin belts, our results suggest that released Merlin undergoes nucleocytoplasmic shutting and mediates export of YAP/TAZ from the nucleus.

A direct assessment of human prion adhered to steel wire using real-time quaking-induced conversion.

Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt-Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 10(2.8) to 10(5.8) SD50. Using this method, we also confirmed that the seeding activities on the wire were removed following treatment with NaOH. As seeding activity closely correlated with the infectivity of prions using the bioassay, this wire-QuIC assay will be useful for the direct evaluation of decontamination methods for human prions.

Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex.

Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 [VprBP] ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 [VprBP] complex, resulting in G1 arrest.