Transcription Factor p63 Regulates Key Genes and Wound Repair in Human Airway Epithelial Basal Cells

Abstract

The airway epithelium in asthma displays altered repair and incomplete barrier formation. Basal cells are the progenitor cells of the airway epithelium, and can repopulate other cell types after injury. We previously reported increased numbers of basal cells expressing the transcription factor p63 in the airway epithelium of patients with asthma. Here we sought to determine the molecular consequences of p63 expression in basal human airway epithelial cells during wound repair. Because at least six isoforms of p63 exist (N-terminally truncated [ΔN] versus transcriptional activation promoter variants and α, β, or γ 3' splice variants), the expression of all isoforms was investigated in primary human airway epithelial cells (pHAECs). We modulated p63 expression, using small interfering RNA (siRNA) and adenoviral constructs to determine the effects of p63 on 21 candidate target genes by RT-PCR, and on repair using a scratch wound assay. We found that basal pHAECs from asthmatic and nonasthmatic donors predominantly expressed the N-terminally truncated p63α variant (ΔNp63α) isoform, with no disease-specific differences in expression. The knockdown of ΔNp63, using specific siRNA, decreased the expression of 11 out of 21 genes associated with epithelial repair and differentiation, including β-catenin, epidermal growth factor receptor, and Jagged1. The loss of ΔNp63 significantly inhibited wound closure (which was associated with the decreased expression of β-catenin and Jagged1), reduced epithelial proliferation as measured by Ki-67 staining, and increased E-cadherin expression, potentially preventing cytokinesis. In conclusion, ΔNp63α is the major isoform expressed in basal pHAECs, and is essential for epithelial wound repair. The role of ΔNp63α in epithelial barrier integrity requires further study to understand its role in health and disease.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium

“…On the basis of these observations, we speculate that in chronic airway diseases including COPD, asthma, and cystic fibrosis, the airways of which show both an increased basal cell population (43)(44)(45) and enhanced expression of EGFR (19,(46)(47)(48)(49), RV may alter the structure and function of airway epithelium and further impair innate defense functions, including barrier function and mucociliary clearance mechanisms. In turn, this may increase the susceptibility to secondary infection by facilitating the crossing of the mucosal barrier by pathogens and their interaction with basolateral receptors, thus altering the cytokine and chemokine responses.…”

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).…”

Supplementing Defect in Club Cell Secretory Protein Attenuates Airway Inflammation in COPD

“…8 Air-liquid interface (ALI) cultures of airway epithelium obtained from endobronchial biopsy specimens (EBBs) collected during bronchoscopy allow new insights into the dynamics and interactions of diff erent epithelial cell phenotypes at steady state and aft er stimulation, in health and disease. [19][20][21][22] We investigated if a CCSP defect is a hallmark of the airway epithelium in COPD and if it could be reproduced ex vivo in ALI cultures. We tried to restore the imbalance between injury and repair by using exogenous CCSP supplementation in ALI cultures obtained from control subjects, smokers, and patients with COPD, and from cultures exposed to cigarette smoke extracts (CSEs), with IL-8 release being the marker of injury.…”