Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway

Tight junctions are the apical cell-cell adhesion that regulate paracellular permeability and are critical for epithelial cell polarity. Molecular architecture of tight junction has been studied extensively, which has confirmed that claudin family of proteins is integral component of tight junction. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential; however, the role of claudin family of proteins play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs and suggest that claudins also participate in cellular contexts other than tight junctions. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. In this review, we have discussed the conceptual framework concerning claudins and their potential implication in cancer. We predict that next several years will likely witness a boom in our understanding of the potential role of claudins in the regulation of tumorigenesis, which may, in turn, provide new approaches for the targeted therapy.

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Claudin Family of Proteins and Cancer: An Overview

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFβ pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

/pdf/novel-dynamics-of-human-mucociliary-differentiation-revealed-1n6xcyvpo9.pdf

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.

Leiomyomas are believed to derive from the transformation of myometrial smooth muscle cells/connective tissue fibroblasts. Although the identity of the molecule(s) that initiate such cellular transformation and orchestrate subsequent growth is still unknown, conventional evidence indicates that ovarian steroids are essential for leiomyoma growth. Ovarian steroid action in their target cell/tissue is mediated in part through local expression of various growth factors, cytokines, and chemokines. These autocrine/paracrine molecules with proinflammatory and profibrotic activities serve as major contributing factors in regulating cellular transformation, cell growth and apoptosis, angiogenesis, cellular hypertrophy, and excess tissue turnover, events central to leiomyoma growth. This review addresses the key regulatory functions of proinflammatory and profibrotic mediators and their molecular mechanisms, downstream signaling that regulates cellular events that result in transformation, and commitments of specific cells into forming a cellular environment with a possible role in development and subsequent growth of leiomyomas.

Proinflammatory and profibrotic mediators: principal effectors of leiomyoma development as a fibrotic disorder.

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airwa...

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair

In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

Rationale: In patients with interstitial lung diseases (ILD) basal cells accumulate within areas of pathological bronchiolization and honeycomb cysts in the peripheral lung. We have previously demonstrated the disease-enriched outgrowth of alveolar basal cells and an anti-fibrotic effect of their secretome in vitro and in vivo. Here, we aimed to characterize similarities and differences between fibrotic alveolar- and airway basal cells. Methods: Alveolar- and airway-derived basal cells were cultured from peripheral lung tissue obtained via transbronchial biopsies (TBB) and from proximal airway via mucosal biopsies (MB) in the same ILD patients. The cells were cultured in an epithelial cell specific growth medium (Cnt-PR-A) and analyzed by single cell RNA sequencing (scRNA-seq), proliferation- and scratch assays, and in an air-liquid-interface (ALI). Results: Both alveolar- and airway basal cells expressed canonical basal cell markers (TP63, KRT5, KRT14, KRT17), and showed similar capacities for wound repair, proliferation and differentiation into mucociliary epithelium. Interestingly, scRNA-seq analysis revealed clear transcriptomic differences between fibrotic alveolar- and airway-derived basal cells. Conclusion: The transcriptomic differences between alveolar basal cells and airway basal cells from lung of ILD patients might be due to the different microenvironment and will be useful to understand the role, function and origin of alveolar basal cells in ILD. Supported by a project grant (310030_192536) by the Swiss National Research Foundation.

Jacqueline Roux

Papers

Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

Characteristics, similarities and differences comparing fibrotic alveolar to airway basal cells

Basal-Like Cells Cultured from Peripheral Fibrotic Lung Tissue Show Transcriptomic Differences to Airway Basal Cells