deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium Ari Jon Arason 1,5 , Hulda R. Jonsdottir 8 , Skarphedinn Halldorsson 4 , Berglind Eva Benediktsdottir 2 , Jon Thor Bergthorsson 1 , Saevar Ingthorsson 1,5 , Olafur Baldursson 6 , Satrajit Sinha 7 , Thorarinn Gudjonsson 1,5 , Magnus K. Magnusson 1,3,5 * 1 Stem Cell Research Unit, Biomedical Center, Faculty of Medicine, University of Iceland, Reykjavik, Iceland, 2 Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland, 3 Department of Pharmacology & Toxicology, Faculty of Medicine, University of Iceland, Reykjavik, Iceland, 4 Center for Systems Biology, University of Iceland, Reykjavik, Iceland, 5 Department of Laboratory Hematology, Landspitali University Hospital, Reykjavik, Iceland, 6 Department of Pulmonary Medicine, Landspitali University Hospital, Reykjavik, Iceland, 7 Department of Biochemistry, Center for Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, United States of America, 8 Institute of Immunobiology, Kantonal Hospital, St. Gallen, Switzerland Abstract The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its DN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the DN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that DNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium. Citation: Arason AJ, Jonsdottir HR, Halldorsson S, Benediktsdottir BE, Bergthorsson JT, et al. (2014) deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium. PLoS ONE 9(2): e88683. doi:10.1371/journal.pone.0088683 Editor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America Received April 16, 2013; Accepted January 9, 2014; Published February 12, 2014 Copyright: ß 2014 Arason et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Icelandic Research Fund (grant number 120423021), Landspitali University Hospital Research Fund, University of Iceland Research Fund and Eimskip University Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: magnuskm@hi.is Introduction Stratified epithelial tissues depend on somatic progenitor cells to maintain their integrity and homeostasis. It has been demonstrated that p63-positive basal epithelial cells serve as a source of other differentiated cells in stratified epithelial tissues including skin (reviewed in [1]). The epithelial layer of the upper airways is lined with a pseudostratified columnar epithelium. A fundamental difference between stratified and pseudostratified epithelia is that all cells of the pseudostratified epithelium are connected to the basement membrane i.e. differentiation does not confer loss of basement membrane adhesion. Deregulation of repair mechanisms and cell differentiation in the bronchial epithelium are important factors in the pathogenesis of several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis and carcinoma [2,3]. Understanding the molecular events regulating hierarchical differentiation and repair mechanisms in airway epithelia is therefore of major interest. p63 expressing basal cells of the mouse trachea and human bronchial tree have been identified as potential progenitor cells during lung development and epithelial repair. This has been shown by different experimental approaches including lineage tracing, injury infliction on the mouse lung and human in vitro culture models [4,5]. Furthermore, p63 has been shown to regulate the expression of cytokeratin (CK) 14 which under normal circumstances is expressed in isolated clusters of basal cells in the bronchial epithelium but is upregulated during airway epithelial repair following injury [5–9]. It has been hypothesized that in the mouse lung, p63 positive basal cells give rise to early progenitor luminal cells positive for CK8 through a notch-dependent mechanism. Later, these cells differentiate terminally to ciliated, clara or goblet cells based on secondary notch signaling processes [10]. Other factors such as the cytokine interleukin 13 (IL-13) have been shown to affect goblet cell differentiation in the airways, with goblet cell hyperplasia reported to be a major contributing factor in diseases like asthma [11,12]. PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88683