deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

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 ΔN 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 ΔN 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 ΔNp63 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.

Low‐level laser irradiation promotes the proliferation and maturation of keratinocytes during epithelial wound repair

“…A previous study showed that rats with a dorsal wound showed faster healing with LLLT (same parameters as utilized herein), which induced enhanced manifestation of young fibroblasts along with a faster closure of the lesions [12]. In agreement with that report, the present study showed that the epithelial tongue cells that migrated towards the wound did indeed express significantly higher amounts of p63, a reliable indicator of epithelial cell proliferation and stratification [28, 29]. …”

“…The anti-p63 antibody was elected for immunohistochemistry due to its close relation to epithelium maturation and stratification, since basal epithelial cells that express p63 serve as a source of differentiating cells from the stratified skin epithelium [28, 29]. …”

Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture

“…In this model, VA10 generates differentiated ciliated cells as well as goblet cells upon induction. 28 In addition, we showed when VA10 is embedded into three-dimensional basement membrane matrix in co-culture with endothelial cells, we see increase in branching structures capturing bronchioalveolar phenotype. 26 Given this plasticity of the basal cell line VA10 and the expression pattern found in IPF lungs, it can be hypothesized that the basal cell could contribute to the fibrotic process in IPF through EMT.…”

“…26 Production of Lentivirus and Cell Transduction Lentiviral transduction was performed as previously described. 28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920) 29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120) 30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems). Transduction of VA10 cells was performed using low MOI volume and 8 mg/ml polybrene and positive cells selected with 0.7 μg/ml puromycin.…”

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

“…However, integrin-a 6 was down-regulated at the level of gene expression ( Figure 5B), suggesting that posttranscriptional regulation may underlie the increased surface expression observed in cells cultured in 3T31Y ( Figure 5A and Figure E8). Expression of the DN P63 isoforms expressed by basal epithelial stem cells (39,40) was not significantly altered by culture conditions ( Figure 5B). In contrast to the tissue-resident airway epithelial cell markers investigated, 3T31Y did not induce expression of pluripotent stem cell markers ( Figure E9).…”