Dysfunctional ErbB2, an EGF receptor family member, in asthmatic airway epithelial cells critically hinders repair after mechanical wounding

Abstract

Background:

Genetic and genomic data increasingly point to the airway epithelium as critical to asthma pathogenesis. Epithelial growth factor (EGF) family members play a fundamental role in epithelial differentiation, proliferation and repair. While ErbB2 mRNA, an EGF family receptor, was reported to be lower in asthma, little is understood about its functional role.

Objective:

To determine whether decreased ErbB2 activation in freshly isolated asthmatic human airway epithelial cells (HAECs) associated with impaired wound closure in vitro.

Methods:

An in vitro scratch wound model of air-liquid interface cultured and freshly isolated HAECs were compared between asthmatic and healthy HAECs in relation to ErbB2.

Results:

Freshly brushed asthmatic HAECs had impaired ErbB2 activation compared to healthy controls. In an in vitro scratch wound model, asthmatic HAECs showed delayed wound closure compared to healthy HAECs. Cell proliferation, assessed by [³H] thymidine incorporation post wounding, as well as expression or activation of ErbB2 and CCND1 at the leading edge of the wound were lower in asthmatic compared to healthy HAECs. A selective ErbB2 tyrosine kinase inhibitor, mubritinib impaired wound closure and decreased CCND1 expression in healthy HAECs, with less impact on asthmatic cells, supporting diminished activity in asthma.

Conclusion:

These results implicate a primary defect in the ErbB2 pathway as constraining asthmatic epithelial repair processes. Restoration of homeostatic ErbB2 function should be considered as a novel asthma therapeutic target.

Capsule summary:

Asthmatic airway epithelial cells have dysregulated wound repair, in association with lower activation of the ErbB2 pathway. Restoration of this pathway might be a novel therapeutic target, potentially restoring a disrupted airway epithelium in asthma.

Introduction

Airway epithelial cells critically maintain barrier function, trigger innate immune pathways and intersect with adaptive immunity (1, 2). Epithelial abnormalities in asthma are increasingly observed (3–6), with recent genomic studies of fresh human airway epithelial cells (HAECs) reporting vast abnormalities in “epithelial growth and repair” related gene expression in association with worsening disease (7, 8). Some studies have suggested epithelial repair abnormalities persist even after long culture duration, supporting genetic (or epigenetic) influences (9). In fact, the most consistently observed genetic locus in relation to asthma, 17q21, is highly enriched for epithelial related genes (10–12), with a recent genome-wide methylation study further linking this locus with molecular pathways and asthma risk (13). However, the precise mechanisms by which these genetic-epigenetic abnormalities impact asthma pathogenesis, including specific alterations in proliferation, migration, apoptosis, tight junction formation, or mucociliary differentiation (and the molecular pathways behind them) remain unclear.

The epidermal growth factor (EGF) receptor (EGFR) family plays a central role in maintaining epithelial cell homeostasis, and EGF itself is necessary for in vitro cell propagation and differentiation (14). The pathway consists of 4 known receptors, erythroblastosis oncogene B 1–4 (ErbB1–4). These receptors, which function as homo-or heterodimers, are activated by a range of EGF family ligands, from EGF, to various “-regulin” family members, to the mucin MUC4β. Phosphorylation of these receptors increases proliferation, migration and tight junction formation, impacts HAEC differentiation, and augments mucus production (15, 16). EGF pathway involvement in asthmatic airway epithelial dysfunction is unclear with some studies suggesting over and others under activity of the pathway (17–20). One of the tyrosine kinase receptors, ErbB2 (also known as HER2, Neu, or CD340), is located within 200 kbp of the 17q12–21 hotspot, and has specifically been identified on genome-wide association study (GWAS) pathway analysis, methylation and gene expression studies in relation to asthma and severe asthma (7, 13, 21). While ErbB2 overactivity is strongly linked to some adenocarcinomas, ErbB2 is also essential for normal HAEC differentiation and integrity. Knockouts are embryonically lethal, confirming its crucial role in survival (22–24), with microarray data reporting reduced ERBB2 mRNA expression in severe asthma clusters (7). However, its role in asthmatic epithelial cell repair has not been investigated.

We hypothesized that the impaired wound repair response observed in asthmatic HAECs both in vivo and in vitro would be caused by a fundamental reduction in proliferative responses to wounding, stemming from decreased activation of ErbB2 pathways. To address this hypothesis, HAECs were analyzed for ErbB2 activation using phosphorylated ErbB2 (pErbB2) expression ex vivo and cultured in an air-liquid interface (ALI) using an in vitro scratch wound model to compare repair and proliferative responses between groups.

Methods

Subjects

Healthy controls and asthmatic subjects were recruited as part of several clinical studies as fully described in the Online Repository. Due to the length of the study, several subgroups of subjects were recruited (see results). This study was approved by the Institutional Review Board of the University of Pittsburgh, and written informed consent was obtained from all study subjects before enrollment.

Bronchoscopy

Bronchoscopy was performed and epithelial brushings obtained from fourth to fifth generation bronchi as previously described (25, 26).

Primary HAEC culture in air-liquid interface

Primary HAECs from the airway brushings were cultured using an air-liquid interface (ALI) (see the Online Repository for details) (25, 26). After 7 days of ALI culture, when full differentiation including ciliated and mucus cells was achieved (27, 28), HAECs were harvested as described below. For the ErbB2 inhibitor experiments, cultured cells were treated with a selective tyrosine kinase inhibitor of ErbB2 (mubritinib, Selleck Chemicals, Houston, TX; 100 nM) or DMSO 16 hours prior to wounding until the time of harvest (29).

Epithelial wound model

To evaluate the wound repair response of airway epithelial cells, a scratch wound model for ALI culture was developed. On day 7 of ALI, three straight lines were manually etched on the cell culture surfaces using a 200 μl pipette tip and the wound area was measured using live cell imaging microscopy (see the Online Repository). The percentage of wound closure (%WC) at 7 hours was reported. Greater %WC indicates better wound healing.

[³H] thymidine incorporation assay

Cell proliferation was measured using the [³H] thymidine incorporation assay on day 7 of ALI as shown in the Online Repository. The time point of 3 hours after wounding was used for baseline [³H] incorporation, and that of 16 hours after wounding was used for measurement of cell proliferation based on the DNA synthesis cycle. The fold change of [³H] thymidine incorporation between 3 and 16 hours after wounding was calculated to compare cell proliferation between healthy and asthmatic HAECs.

Quantitative reverse transcription PCR (qRT-PCR) and western blot analysis

On day 7 of ALI, and 7 hours after scrape wounding, HAECs were harvested and processed for qRT-PCR and western blot analysis of targeted cyclin D1 (CCND1, as a cell proliferation marker regulated by activation of EGF and other pathways(30, 31)) as well as ErbB2. Freshly brushed HAECs were also processed for total and phosphorylated ErbB2 protein expression. Detailed methods were described in the Online Repository.

Immunofluorescent staining

At 7 hour post scraping, the entire Transwell membrane was processed for immunofluorescent staining for CCND1 and phosphorylated ErbB2 (pErbB2). Cytospin samples of freshly brushed HAECs were also stained for pErbB2. For Transwell membrane samples, regions of interest (ROIs) were randomly and intermittently obtained along wound leading edge on each wounded culture. The numbers of CCND1 positive cells and the fold-change of fluorescent intensity of phosphorylated ErbB2 between the wound leading edge and the non-wound area were measured. For cytospin samples, the percentages of pErbB2 positive cells were analyzed (see the Online Repository).

Statistics

Summary statistics are presented as mean ± SEM or median (IQR) for fractional exhaled nitric oxide (FeNO), in vivo pErbB2 expression analysis, and the fold-change of [³H] thymidine incorporation between 3 and 16 hours due to non-linear distribution of the data. In vitro comparisons across subject type or condition are expressed as fold-change compared to control, primarily because of varying baselines. The differences in the fold-changes are then analyzed by ANOVA, followed by multiple comparison testing with Bonferroni correction (P value < 0.0125). All Statistical analysis was performed with JMP Pro software (version 12, SAS Institute, Cary, NC). P values of less than 0.05 were considered statistically significant.

Results

Study subjects

Due to the complexity of the study and sample availability, each experiment used subgroups of the overall study population (Table 1, and Tables E1a–e in the Online Repository). There were no demographic differences between subgroups except for the ErbB2 inhibitor experiment which included no severe asthmatics due to ease of availability and the observed lack of impact of severity on wound closure.

Table 1.

Subject demographics

	in vivo (freshly brushed HAECs)		in vitro (ALI culture)
Experiment	WB	IF	WC/mRNA/ WB	[3H] thymidine	ErbB2 inhibitor (WC/WB/IF)	P value
N	26	13	34	13	12
HC/MMA/SA	6 / 8 / 12	4 / 5 / 4	12 / 11 / 11	5 / 4 / 4	6 / 6 / 0
Age, years	38.7 ± 14.1	38.5 ± 12.2	42.1 ± 11.2	46.8 ± 8.2	33.9 ± 13.3	0.08
Sex, Male/female	9 / 17	5 / 8	13 / 21	5 / 8	6 / 6	0.93
Race, C/AA/O	20 / 3 / 3	11 / 1 / 1	25 / 8 / 1	10 / 3 / 0	10 / 2 / 0	0.62
Inhaled corticosteroids, yes/no	16 / 10	7 / 6	16 / 18	7 / 6	3 / 9	0.32
FEV1, %predicted	71.5 ± 23.1	73.2 ± 31.2	79.6 ± 26.2	84.6 ± 24.9	91.0 ± 17.0	0.18
FeNO, ppb	26.5 (19.7 – 44.3)	16 (14.5 – 31.5)	24 (17 – 64)	23 (17.3 – 60.3)	16 (10.5 – 35.7)	0.27

Data are represented as mean ± SD or median (IQR). P values were analyzed by ANOVA.

Abbreviations: AA, African American; ALI, Air-liquid interface; C, Caucasian; FeNO, Fractional exhaled nitric oxide; FEV₁, Forced expiratory volume in 1 second; [³H] thymidine, [³H] thymidine incorporation assay; HAECs, Human airway epithelial cells; HC, Healthy controls; IF, Immunofluorescent staining; MMA, Mild-to-moderate asthmatics; O, Others; SA, Severe asthmatics; WB, Western blotting; WC, Wound closure.

pErbB2 expression is lower in freshly isolated asthmatic HAECs than in HAECs from healthy subjects

Total ErbB2 (tErbB2) and pErbB2 protein expression was analyzed in freshly isolated HAECs (Table 1 and Table E1a in the Online Repository) (7). While there were no differences in tErbB2 expression, pErbB2 (and the ratio to total ErbB2 [p/t ErbB2]) was lower in asthmatic HAECs than HC HAECs (Fig. 1A–C and G). There were no differences by asthma severity (Fig. 1D–F). To confirm these results, pErbB2 expression was evaluated by immunofluorescent staining on freshly brushed HAEC cytospins (Table E1b in the Online Repository). Asthmatic HAECs had fewer pErbB2 positive cells than HC HAECs (Fig. 1H–I), without difference by severity.

Fig. 1. in vivo phosphorylated ErbB2 protein expression in freshly isolated HAECs is lower in asthmatic than in healthy subjects.

Total and phosphorylated ErbB2 (tErbB2 and pErbB2) protein expression in freshly isolated HAECs was analyzed by western blotting (A-G) and immunofluorescent staining of cytospin samples (H-I). (A, D) There were no differences in tErbB2 protein expression across the groups. (B, E) pErbB2 protein and (C, F) the ratio of phosphorylated to total ErbB2 (p/t ErbB2) was lower in asthmatic HAECs, but without difference by severity. (G) Representative western blots for ErbB2 among freshly isolated HAECs. (H) Representative immunofluorescent pictures for pErbB2 among cytospin HAECs samples. Green: pErbB2, Blue: nucleus. The white bar represents 100 μm. (I) pErbB2 positive cells were less in asthmatic HAECs than in healthy HAECs. Each bar represents median with interquartile range. Statistical analysis was performed by Wilcoxon rank sum test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma. Abbreviations: HC, Healthy control; MMA, Mild-to-moderate asthma; SA, Severe asthma.

Asthmatic HAECs exhibit abnormal wound closure in a scratch wound model

Among the subgroup of in vitro wound-closure experiment, there were lower percent predicted values of FEV₁ and higher FeNO levels in asthma subjects as compared to HCs, while age, sex, and race were not different (Table E1c in the Online Repository). Seven hours after scraping (day 7 of ALI culture), %WC was 51.6±4.0% in HCs compared to 38.5±2.4% in asthmatic HAECs (mean±SEM; P = 0.01), as measured by live cell microscopy (Fig. 2A). There were no differences in %WC between MMA and SA cells (37.1±3.4% and 39.8±3.6%; P = 0.59). However, asthmatic patients in the lowest quartile for %WC (%WC <30%) had higher FeNO levels than those in the highest %WC quartile (%WC >45%) (80.0±24.1 ppb vs 31.4±11.8 ppb; P = 0.006, Fig. 2B). No other clinical characteristics (including inhaled corticosteroid use, atopic status, exacerbation history, physiologic or inflammatory patterns) differentiated the %WC in asthmatics or HCs.

Fig. 2. Wound closure and cell proliferation after wounding is impaired in asthmatic HAECs.

(A) Wound closure was decreased in asthmatic HAECs compared to healthy HAECs in air-liquid interface (ALI) culture. HAECs were cultured in ALI for 7 days. Wound area was measured at baseline and 7 hours after wounding, and percentage of wound closure at 7 hours after wounding was calculated. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. (B) Asthmatic patients with the worst HAEC wound closure (WC) (bottom quartile) had higher FeNO than those whose wound closure overlapped with normal wound repair (top quartile). Hi-%WC was defined as %WC >45%, and Lo-%WC was defined as %WC <30%. (C) Cell proliferation after wounding was impaired in asthmatic HAECs compared to healthy HAECs. [³H] thymidine incorporation at 16 hours after wounding was higher in healthy HAECs than in asthmatic HAECs. The fold-change of [³H] thymidine incorporation between 3 to 16 hours after wounding was significantly higher in healthy HAECs than in asthmatic HAECs. Statistical analyses were performed by Student’s t test and Wilcoxon rank sum test, respectively. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Cell proliferation is impaired in asthmatic HAEC after wounding

[³H] thymidine incorporation was measured to evaluate cell proliferation after wounding. While there were no differences in proliferation as measured by counts per minute (CPM) at 3 hours post wounding, by 16 hours the [³H] thymidine CPM were significantly higher in HCs than in asthmatic HAECs (Fig. 2C). The fold change of [³H] thymidine incorporation between 3 and 16 hours was also less in asthmatic compared to HC HAECs [medians (IQR) of 1.56 (1.29 – 1.95) and 2.04 (1.84 – 8.05); P = 0.03, Fig. 2C]. Asthma severity did not impact [³H] thymidine incorporation (P = 0.15). No significant increase was observed at 7 hours post wounding (Fig. E1 in the Online Repository). Despite similar trends in wound repair and proliferation, [³H] thymidine incorporation and %WC did not correlate.

CCND1 expression after wounding is decreased in asthmatic HAEC

CCND1 expression, in the total cells from the transwell membrane, increased compared to control conditions at both mRNA and protein levels in all cultures after wounding (Figs. 3A, 3B, and Fig. E2 in the Online Repository). Wound-related fold induction of CCND1 protein (but not mRNA) was greater in HC than in asthmatic HAECs (Figs. 3B and 3C). Similarly, expression of CCND1 at the wound edge, evaluated by immunofluorescent staining, revealed greater CCND1 positive cells at the leading edge of the wound in HC compared to asthmatic HAECs (Figs. 3D and 3E). CCND1 expression values did not correlate with %WC.

Fig. 3. Wounding induced CCND1 expression is reduced in asthmatic HAECs.

(A) CCND1 mRNA expression. (B) CCND1protein expression. (C) Representative western blot for ErbB2 and CCND1 protein expression with or without wounding. (D) Representative immunofluorescent staining images for CCND1 at wound leading edge. Red: CCND1, Blue: nucleus. The white bar represents 300 μm. (E) CCND1 positive cells along wound leading edge were higher in healthy as compared to asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Phosphorylation of ErbB2 along the leading edge of the wound is decreased in asthmatic HAECs

To determine whether ErbB2 pathway activation could be contributing to the abnormal wound repair, we evaluated ERBB2 mRNA expression in HAECs at 7 hours. ERBB2 mRNA did not change following wounding, nor were there differences between subject groups when evaluating the entire transwells (Fig. 4A). However, despite the lack of significant changes, ERBB2 mRNA correlated strongly with CCND1 mRNA following wounding (ρ=0.82, p<0.0001). In western blot, two bands were observed for pErbB2: a full length (transmembrane form, 185kDa) and a truncated form (nuclear translocated form, 120kDa), as shown in Fig. 3C and 5C. The former one was used for analyses. While there were no consistent changes in pErbB2 or tErbB2 protein after wounding from either subject group by western blot (Figs. 4B, 4C, and Fig. E3 in the Online Repository), the ratio of phosphorylated to total ErbB2 protein (p/t ErbB2) was less in asthmatic compared to HC HAEC wounds (Fig. 4D). In small numbers, there were no other correlations between ErbB2 expression and any clinical parameters including FEV₁ or dose of inhaled corticosteroids. Local ErbB2 expression at the leading edge of the wound was also analyzed by immunofluorescence 7 hours after wounding. pErbB2 was upregulated along the leading edge of the wound compared to the non-wounded area in the intensity profile analysis (Fig. 4E). The fold change in the intensity of pErbB2 from the “no wound/control” area to the leading edge of the wound was less in asthmatic than in HC HAECs (Fig. 4F), supporting lower wound associated activation of ErbB2 in asthma. ErbB2 expression did not directly correlate with %WC.

Fig. 4. Phosphorylation of ErbB2 after wounding is impaired in asthmatic HAECs.

(A) ERBB2 mRNA expression. (B) Total ErbB2 protein expression. (C) Phosphorylated ErbB2 protein expression. (D) Ratio of phosphorylated to total ErbB2 protein (P = 0.09 and 0.04 for difference in ratio between HC and asthma before and after wounding, respectively). (E) Representative immunofluorescent staining pictures and their intensity profile for phosphorylated ErbB2 and F-actin at wound leading edge. Red: phosphorylated ErbB2, Green: F-actin, Blue: nucleus. The white bar represents 100 μm. (F) Phosphorylated ErbB2 expression along leading edge of the wound was higher in healthy as compared to asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test. Symbols; ○: healthy control, ▲: mild-to-moderate asthma, ■: severe asthma.

Fig. 5. Inhibition of ErbB2 phosphorylation impairs wound closure and the effect is greater in HC HAECs.

(A) Mubritinib (100nM) treatment decreased wound closure after 7 hours of wounding with greater impact in healthy HAECs as compared to asthmatic HAECs (P = 0.04 for difference in decrease in wound closure between HCs and asthma). (B) CCND1 protein expression was inhibited by mubritinib treatment in healthy HAECs, but not in asthmatic HAECs (P = 0.04 for difference between HC and asthma). (C) Representative western blot for ErbB2 and CCND1 protein expression with or without mubritinib treatment. (D) Phosphorylation of ErbB2 along leading edge of the wound was impaired by mubritinib primarily in healthy HAECs. Representative immunofluorescent staining for phosphorylated ErbB2 and F-actin at leading edge of the wound. Red: phosphorylated ErbB2, Green: F-actin, Blue: nucleus. The white bar represents 100 μm. (E) Intensity ratio of phosphorylated ErbB2 to F-actin at wound leading edge was decreased by mubritinib in healthy HAECs, not in asthmatic HAECs. Each bar represents mean ± SEM. Statistical analysis was performed by Student’s t test.

Inhibition of ErbB2 phosphorylation differentially impacts wound closure in HC compared to asthmatic HAEC

Inhibition of ErbB2 phosphorylation by mubritinib significantly decreased wound closure in both HC and asthmatic HAECs (Fig. 5A). However, the decrease in wound closure following mubritinib treatment was greater in HC than in asthmatic HAECs (Fig. 5A). Similarly, CCND1 protein expression decreased with mubritinib treatment to a greater degree in HC than in asthmatic HAECs (Fig. 5B and C). These results were verified using immunofluorescent staining, as mubritinib treatment suppressed ErbB2 phosphorylation along the leading edge to a greater degree in HC than in asthmatic HAECs, likely due to lower baseline ErbB2 activation in the asthmatic cells (Figs. 5D and 5E).

Discussion

In this study, lower ErbB2 activation was observed in freshly brushed asthmatic HAECs compared to cells from HCs. These ex vivo findings were then mechanistically explored in primary ALI culture, where asthmatic HAECs exhibited diminished wound closure, a reduction in proliferation, changes in CCND1 expression and ErbB2 activation as compared to HC cells. Combined with previous genetic and genomic studies, these results strongly support abnormalities of the ErbB2 pathway in human asthmatic airway epithelial cells upon wound repair.

The delayed airway epithelial repair in asthma observed here could allow easier access of pathogens to the submucosa and the rich array of immune cells located there, promoting airway inflammation or even airway remodeling. The reasons for the low activation of the ErbB2 pathway in asthmatic HAECs observed are not well known. While genetic factors controlling both expression and possibly activity of the ErbB2 pathway are likely, given the gene’s strong linkage disequilibrium with the 17q12–21 “hotspot”, epigenetic factors could also be involved (10–13). Our findings that the worst wound repair was seen in cells from patients with higher FeNO levels (in vivo) suggest that T2 inflammation may have epigenetically altered the cells in such a way as to promote lower ErbB2 activation responses even after nearly a month in culture. Alternatively, genetic or epigenetic changes in the HAECs could promote generation of the T2 immune processes observed in vivo. In the previous microarray study, reduced ErbB2 expression was seen in the patients with the highest FeNO, a marker of T2 inflammation (7). IL-13 has been reported to have epigenetic effects through inhibition of microRNA expression, which may regulate cell differentiation, at least in part through effects on the NOTCH1 pathway. However, its ability to impact ErbB2 expression/activation is unknown (32). Finally, loss of EGF receptor family ligands including MUC4β, the only known endogenous ligand for ErbB2 homodimers, could also prolong epithelial repair, through decreased activation of heterodimers of ErbB2 with other EGFR family members (33, 34).

Although many pathways are considered to be involved with epithelial repair including migration, apoptosis and barrier function, our studies suggest that reduced proliferation is playing an important role in asthmatic HAECs. ErbB2 is widely considered critical to wound healing and epithelial differentiation, but the vast majority of published studies address ErbB2 in the context of cancer including breast and gastric tumors, where its overexpression is believed to contribute to its dimerization with EGFR or ErbB3 resulting in aberrant cell survival and proliferation (35–37). However, ErbB2 has also been studied in relation to epithelial repair and differentiation. Vermeer et al. identified ErbB2 phosphorylation along the leading edge of a physical wound using an ALI model of HAECs (38). They reported that treatment with an anti-ErbB2 antibody decreased ciliated and increased metaplastic cells in differentiated HAEC culture (22). The findings reported here, of very low expression (and activity) in freshly brushed cells (by mRNA and protein) and low activity in cultured asthmatic cells following wounding, support the polar opposite situation in which low levels of ErbB2 activity and expression, prevent normal epithelial repair, and may explain why Erbb2 knock-out mice are embryonically lethal (24).

To confirm the relationship between wound closure, CCND1 and ErbB2, we inhibited the pathway using mubritinib, a selective HER2 tyrosine kinase inhibitor. Mubritinib is a selective ErbB2 inhibitor, compared with several other tyrosine kinases which nonselectively also target EGFR, FGFR, and PDGFR (29). Mubritinib’s anti-proliferative effect was only seen in an ErbB2-overexpressing breast cancer cell line (IC₅₀ of 5 nM), without inhibitory effects in EGFR/ErbB1-overexpressing cell lines (IC₅₀ of >25 μM). As anticipated, inhibition of ErbB2 by mubritinib decreased ErbB2 phosphorylation, most notably at the leading edge of the wound, while it profoundly decreased CCND1 mRNA and protein, supporting the link between this specific EGF family receptor and cell proliferation. Importantly, these effects were significantly greater in HCs where the expression and activation of ErbB2 was highest, having minimal effect in the asthmatic (ErbB2 suppressed) cells.

ERBB2 and CCND1 mRNA expression was highly correlated in cultured ALI cells, suggesting interactions between ErbB2 and CCND1 in cell proliferation. This correlation is consistent with our previous published gene expression study (7). Although other factors could also stimulate proliferation in epithelial cells, the EGF pathway is widely considered the most relevant (39). Importantly, our published gene array data did not show differences in expression of any other EGF family receptors between HC and asthmatic HAECs. These findings contrast with a recent RNA-Seq profiling study of sputum-derived cells which reported concomitant upregulation of Type-2 and EGFR/ERBB1 and ERBB2 in house dust mite sensitive wheezers than nonatopic controls. However, they did not control for cell type, with these differences potentially due to more epithelial cells in asthmatic sputum (40). Given the complexities of dimerization patterns among the EGF receptors and its impact on ligand selectivity, however, it is conceivable other EGF receptors are involved to some degree as well.

Our studies have both similarities and differences with previous studies. Stevens et al. also reported a defect in wound repair but in airway epithelial cells in a monolayer system, and from children (41). This system is very different from the differentiated/polarized cells studied here. An additional study showed that over expression of plasminogen activator inhibitor-1 (PAI-1) which is induced by EGF contributed to the abnormal repair in asthmatic HAECs (42). Their findings contrast with ours, perhaps due to differences in the culture systems.

There are some limitations in this study. First, the sample size for the in vitro studies is small. However, the sample size of this study was much larger than any similar studies and the diagnosis of asthma was rigorously confirmed. Second, tErbB2 protein by western blot did not differ between asthmatic and HC cells in vitro. However, phosphorylation is more critical to evaluate ErbB2 pathway activation than total ErbB2 expression as their homo or hetero dimerization with other EGFR family members is necessary for pathway activation (43), and importantly we observed markedly lower ErbB2 phosphorylation both in vitro, in response to wounding and in vivo in freshly brushed, perhaps already “wounded” asthmatic epithelial cells. Third, there was discordance in the time points between [³H] thymidine which was measured between 3–16 hours and %WC after 7 hours. %WC was evaluated at 7 hours as most wounds were completely closed at 16 hours. Proliferation signals could continue beyond the simple closure, with the 3–16 hour window capturing the total amount. However, it is almost certain that other wound healing processes, like migration, also contribute wound closure, particularly at the earlier timepoints. Fourth, there was no difference by asthma severity in the wound closure experiments. This could certainly be due to the prolonged (~4 weeks) de-differentiation and re-differentiation of the cultured cells and differences in the overall environment of the cultured cells vs those in the asthmatic epithelium. Finally, current technical limitations of primary HAECs prevent us from adding or generating specific stimuli to phosphorylate ErbB2 in asthmatic HAECs, preventing us from directly confirming the importance of the deficiency of this ErbB2 pathway in asthmatic cells.

In conclusion, decreased activation of ErbB2 impedes the proliferative response to wound repair in both in vitro HAEC models and in freshly brushed HAECs in vivo. The relationship between the in vitro and in vivo systems, the impact of ErbB2 on the CCND1 pathway and wound repair, combined with the previous genetic data all suggest that studies which focus on understanding the reasons for the downregulation of this pathway may lead to effective and even preventive asthma therapies, reversing a fundamental underlying “defect” of the disease.

Key Messages:

• Asthmatic airway epithelial cells have impaired wound closure with decreased cell proliferation after wounding compared to healthy controls

• Dysregulated activation of ErbB2 was observed both in in vivo and in vitro among asthmatic airway epithelial cells, suggesting a novel therapeutic target for asthma treatment

Abbreviations:

HAECs

human airway epithelial cells

EGFR

epidermal growth factor receptor

ErbB2

erythroblastosis oncogene B2

pErbB2

phosphorylated ErbB2

tErbB2

total ErbB2

GWAS

genome-wide association study

ALI

air-liquid interface

HCs

healthy controls

FEV₁

forced expiratory volume in 1 second

%WC

percentage of wound closure

CCND1

cyclin D1

ROIs

regions of interest

FeNO

fractional exhaled nitric oxide

MMA

mild-to-moderate asthma

SA

severe asthma

UPMC

The University of Pittsburgh Medical Center