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.

Figure 1. ΔNp63 is expressed in bronchial basal cells in situ and is a major p63 isoform in basal cells in vitro.

ΔNp63 is expressed in basal cells in normal human bronchi (a). VA10 cells cultured in ALI express ΔNp63 at the basolateral side and not on the apical side (b). ΔNp63 shows 167,5 fold expression compared to TAp63 in bronchial basal cell line VA10, as calculated by ΔΔct obtained by qRT-PCR. GAPDH was used as endogenous control. (c). Scale bars 50 µm. ***p≤0.001.

Figure 2. Knockdown of p63 affects cellular morphology, proliferation and migration.

Western blot showing lentiviral siRNA knockdown of p63 in VA10 (a). A subset of VA10^p63kd (KD) cells obtains an elongated morphology in monolayer culture (b). A proliferation assay reveals 40% decrease in proliferation of KD cells compared to scrambled. Error bars represent 95% confidence intervals (c). A transwell migration assay shows reduced ability of KD cells to migrate through the filter (d). ***p≤0.001, **p≤0.01.

Figure 3. p63 is necessary for a quick wound healing response.

Wound healing assay shows decreased healing capacity of VA10^p63kd cells (right) compared to VA10^Scr cells (left) after 1,3 and 6 hours (a). Images represent results obtained from 4 independent experiments. Graph showing relative wound healing speed between VA10^p63kd cells and VA10^Scr cells (b). ***p≤0.001, **p≤0.01, *p≤.0.5.

Figure 4. VA10^p63kd cells lose survival ability.

VA10^p63kd (KD) cells enter senescence when cultured for prolonged period in monolayer, as shown with β-galactosidase staining (a). To quantify the senescence the pixel intensity is represented in bars for VA10^Scr compared to VA10^p63kd. Scale bars 50 µm. *p≤.0.5.

Figure 5. VA10^p63kd form impaired lung epithelium in ALI culture.

VA10^p63kd cells form simple epithelium with random epithelial budding (7 days after initiation of ALI) (a) and significant downregulation of CK14 (14 days after initiation of ALI) (b). The VA10^p63kd epithelium does not form transepithelial electrical resistance (c) and has high permeability of Flu-Na (d) compared to scrambled control (14 days after initiation of ALI). Scale bars 50 µm. ***p≤0.001.

Figure 6. p63 is necessary for IL-13 induced goblet cell differentiation in ALI culture.

A subset of VA10^Scr differentiates to goblet cells when stimulated with IL-13 (left panel). VA10^p63kd cells are unable to form goblet cells when stimulated with IL-13 (right panel). Scale bars 50 µm. **p≤0.01.