Background
Asthma is a chronic inflammatory disorder of the airways characterized by inflammation, airway hyperresponsiveness, and reversible airflow obstruction [
1]. Several cell types have been implicated in the pathogenesis of asthma; airway epithelial barrier dysfunction plays an important role [
2]. Therefore, epithelial barrier function, which biologically limits the passage of foreign substances, including inhaled allergens, into the body, plays an important role in airway defense. In general, epithelial barrier function is maintained through a series of cell junctions on the apical side of cells, including tight junctions (TJs) and adherence junctions (AJs) [
3].
Bronchial hyperresponsiveness (BHR), the key feature of asthma, is a functional abnormality in which airway constriction is triggered by environmental stimuli that otherwise do not affect healthy individuals. Although airway inflammation is strongly implicated in BHR, the mechanisms underlying BHR remain unclear [
4,
5]. Protocadherin-1 (
PCDH1) was recently identified as a susceptibility gene in asthma [
6,
7]. Koppelman et al. performed linkage and mapping analysis in 200 Dutch asthmatic patients with the goal of detecting genes on chromosome 5q31–q33 that are associated with BHR [
6]. They found a significant relationship between
PCDH1 and BHR. A follow-up study revealed the same significant relationship between
PCDH1 and BHR in seven of eight populations analyzed (Dutch, English, and American subjects) [
6].
PCDH1 belongs to the cadherin protein superfamily and contains a 110-amino acid repeat sequence called the cadherin motif. The cadherin superfamily includes E-cadherin (E-cad), N-cadherin, P-cadherin, desmosomal cadherin, and PCDH [
8]. Koning et al. found that
PCDH1 mRNA expression increased during differentiation of cultured airway epithelial cells, which suggested that PCDH1 is important in this process [
9].
Formation of the epithelial barrier is an important process during airway epithelial differentiation; however, it is not clear if PCDH1 participates in epithelial barrier formation. In this study, we tested the hypothesis that functional abnormalities due to PCDH1 dysregulation may affect epithelial barrier formation and thereby contribute to the pathogenesis of asthma.
Methods
Cells and reagents
Transformed human bronchial epithelial cells (16HBE14
0−, abbreviated as 16HBE cells [
10,
11] and 1HAE
0−, abbreviated as 1HAE cells [
12]) were kindly provided by Prof. Dieter C. Gruenert (Gene Therapy Center, Cardiovascular Research Institute, Department of Laboratory Medicine, University of California). Calu-3 cells, an airway epithelial cell line derived from lung cancer, were obtained from the American Type Culture Collection (Rockville, MD, USA) [
13]. Dexamethasone (Dex) and fluorescein isothiocyanate-labeled dextran (FITC-dextran; 4 and 10 kDa) were purchased from Sigma Chemical Company (St. Louis, MO, USA).
Cell culture
16HBE cells were grown in minimum essential medium (MEM) with 10 % (v/v) fetal bovine serum (FBS). For our experiments, these cells were passaged 20–40 times. Calu-3 cells were maintained in a 1:1 mixture of Ham’s F12 (Gibco Invitrogen Corp., Paisley, UK) and Dulbecco’s Modified Eagle Medium (Sigma), with 10 % FBS (SAFC Biosciences, Lenexa, KS, USA), and passaged 20–40 times before use. 1HAE cells were grown in MEM with 10 % (v/v) FBS and passaged 10–30 times before use.
siRNA transfection
16HBE cells were grown in six-well plates to 50 % confluence and transfected individually with either 50-nM Silencer Select Control small interfering RNA (siCtlRNA, cat. 12935–112; Invitrogen, Carlsbad, CA, USA) or human PCDH1 siRNAs (siPCDH1_1, siPCDH1_2, and siPCDH1_3, all obtained from Sigma-Aldrich) for 24 h using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. The transfected cells were seeded on Transwell chambers (Corning Life Sciences, Corning, NY, USA) before replacing the transfection medium with complete medium with or without Dex.
RNA extraction and real-time PCR
Total RNA was extracted from 16HBE cells with the RNAiso Reagent (TaKaRa, Japan). First-strand cDNA was synthesized from 2 μg total cellular RNA with the PrimeScript RT reagent Kit (TaKaRa). To amplify PCDH1, specific primers were designed based on the gene sequences. Gene-specific primer sets were designed for human PCDH1 isoforms 1 and 2 as follows: PCDH1 isoform 1, 5′-GACTCTTCCAGATTGGGTCACAT-3′ and 5′-CTTGCCGCGGTCACTGA-3′; PCDH1 isoform 2, 5′-TGCCAATGCAGAAATCGAATAC-3′ and 5′-CGGGCCCTGAACAGTGAT-3′. Primers for amplification of GAPDH were used as an internal control: 5′-CAAGTTCAACGGCACAGTCAAG-3′ and 5′-ACATACTCAGCACCAGCATCAC-3′. The Applied Biosystems 7300 Fast Real-Time PCR System with SYBR green PCR master mix (Applied Biosystems) were used according to manufacturer protocols. The reactions were incubated in a 96-well optical plate at 95 °C for 20 s, followed by 40 cycles each of 95 °C for 3 s and 60 °C for 30 s. The threshold cycle (Ct) data were obtained using default threshold settings. Ct is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
Measurement of transepithelial electrical resistance
16HBE and 1HAE cells were seeded onto Transwell inserts (Costar, New York, NY, USA) at a density of 2 × 105 cells/cm2. Calu-3 cells were seeded onto Transwell inserts at a density of 1 × 105 cells/cm2. Cell layer integrity was evaluated by measuring transepithelial electrical resistance (TER) with Millicell-ERS equipment (Millipore Co., Bedford, MA, USA).
The permeability of cell monolayers was determined by FITC-dextran fluxes across the cell layer. A solution containing FITC-dextran of 4 or 10 kDa (1 mg/ml) was added to the apical compartment. Samples (100 μl) were removed from the basal compartments 60 min after addition of FITC-dextran and measured by a PTI fluorometer at 492 nm (excitation) and 520 nm (emission).
Apoptosis assay
The Annexin V/FITC and propidium iodide (PI) apoptosis detection kit (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to quantitatively measure the phosphatidylserine in apoptotic cells. Briefly, transfected cells (siCtl, siPCDH1) (5 × 10
5 per well) were seeded into 6-well plates. After 24 h, the cells were harvested and washed three times with ice-cold phosphate-buffered saline (PBS) (pH 7.2). After washing, each sample was centrifuged at 1300 rpm for 3 min at 4 °C. Annexin V/FITC and PI double-staining were performed according to manufacturer instructions. Apoptosis was analyzed on a FACScan flow cytometer (Becton-Dickinson, Heidelberg, Germany) and Annexin V-positive, PI-negative cells were scored as apoptotic (Fig
4). Double-stained cells were considered to be necrotic or late apoptotic.
Immunofluorescence microscopy
After the indicated culture period on Transwells, cells were fixed with 4 % paraformaldehyde for 30 min at 37 °C. Anti-human ZO-1 mAb (Zymed Laboratories Inc., San Francisco, CA), anti-human E-cad rabbit mAb (Cell Signaling Technology, MA), or anti-human PCDH1 mAb (Santa Cruz) was used as a primary antibody, and Alexa 488-conjugated anti-mouse IgG was used as a secondary antibody. An FV1000-D laser scanning confocal microscope with a 60× objective lens was used to investigate expression.
Patients
Sixteen patients with chronic rhinosinusitis (CRS) and nine asthmatic patients were enrolled. Tissue samples were obtained during surgical biopsies from the patients. CRS and asthma were defined by the criteria established by the American Academy of Otolaryngology–Head and Neck Surgery Chronic Rhinosinusitis Task Force [
14] and the Global Initiative for Asthma guidelines [
1], respectively. The subjects’ clinical characteristics are shown in Tables
1 and
2. All ethmoid sinus tissues were collected during surgery to remove nasal polyps. We used nasal tissues from the patients who underwent surgery for nasal septal deviation as a control for CRS. Five lung tissue samples from patients with asthma were collected during surgery for pneumothorax, one sample was collected by pneumonectomy for lung cancer, and three samples were collected during autopsy. We used lung tissues from the patients who underwent surgery for pneumothorax or lung cancer as a control for asthma. The study was approved by the Nihon University Itabashi Hospital Ethics Committee, and written informed consent was obtained from all patients.
Table 1
Clinical characteristics of patients with chronic rhinosinusitis (CRS)
Sex (M/F) | 6/3 | 10/6 | |
Age (years), median (range) | 57.7 (19–82) | 57 (24–70) | 0.145073 |
Blood eosinophil (/μl)a | 122 | 345 | 0.030078 |
Smoking (%) | 44.4 | 33.3 | |
Table 2
Clinical characteristics of patients with asthma
Sex (M/F) | 9/0 | 8/1 | |
Age (years), median (range) | 34.8 | 45.6 | 0.114272 |
FEV1.0 (% predicted)a | | 59.3 | |
FVC (% predicted)a | | 65.1 | |
Blood eosinophil (/μl)a | 173 | 518 | 0.006926 |
Smoking (%) | 66.6 | 33.3 | |
Western blotting
Stimulated cells were washed twice with ice-cold PBS and lysed in Tris-buffered saline containing 1 % Nonidet P-40, 60 mM octyl-β-glucoside, 2 mM phenylmethylsulfonylfluoride, 10 μg/ml aprotinin, 2 μg/ml leupeptin and pepstatin A, 50 mM NaF, and 1 mM sodium orthovanadate for 30 min on ice. The lysates or immunoprecipitates were centrifuged for 15 min at 14000
g. The samples for polyacrylamide gel electrophoresis (PAGE) analysis were mixed with 4× XT sample buffer (Bio-Rad, Hercules, CA) and boiled for 4 min and separated on 10 % sodium dodecylsulfate-PAGE and transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with anti-human ZO-1 mAb (Zymed), anti-human OCLN rabbit mAb (Zymed), anti-human E-cad rabbit mAb (Cell Signaling), and anti-human PCDH1 mAb (Santa Cruz) as a primary antibody and an appropriate secondary horseradish peroxidase-conjugated antibody (Fig.
6). Signals were detected using enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).
Immunohistochemistry
We focused on PCDH1 expression in ciliated airway epithelial cells (CECs) from the noninflamed region (NR), where there are few infiltrated inflammatory cells and CECs are histologically intact. We also examined inflamed regions (IR) where inflammatory cells such as eosinophils and lymphocytes had infiltrated the submucosa and where histology indicated that the CECs had sustained damage such as partially shed epithelium or separation of cell junctions. The lung and nasal tissues were paraffin embedded and then cut into sections. These sections were deparaffinized and rehydrated. After antigen retrieval, the endogenous peroxidase was inactivated by 3 % hydrogen peroxide in methanol. Then, these sections were incubated with the primary antibody against PCDH1 (1:500) at room temperature for 1 h for 30 °C at room temperature. Staining was performed with 3,3′-diaminobenzidine, and counterstaining was performed using hematoxylin.
Sections were dehydrated in absolute ethanol and dehydrated in an absolute ethanol series and xylene. After mounting, the sections were observed by light microscopy. The primary antibody was replaced with phosphate-buffered saline (PBS) in the negative controls. All sections were scored in a semiquantitative manner by considering the intensity of cell staining. Intensities were classified as 0 (no staining), +1 (weak staining), +2 (distinct staining), and +3 (very strong staining).
Statistical analysis
Normally distributed data were expressed as means ± standard errors, and differences between groups were analyzed by Student’s t-test. Where not normally distributed, data were summarized using the median and interquartile range and were evaluated by nonparametric Wilcoxon rank sum or Mann–Whitney U test. All data were analyzed by Prism (GraphPad Software, La Jolla, CA).
Discussion
To our knowledge, this study is the first to demonstrate that
PCDH1, which has been identified as an airway hyperreactivity-susceptibility gene, has an important role in the formation and maintenance of the intercellular junctions that comprise the airway epithelial barrier. Epithelial barrier dysfunction contributes to the pathogenesis and development of bronchial asthma and CRS [
18]. Our study suggests that reduced PCDH1 expression and function is related to the pathogenesis of allergic airway inflammation in bronchial asthma and CRS.
Epithelial barrier function is maintained by TJs and AJs. AJ formation is considered to be especially important in epithelial polarization, which in turn facilitates TJ formation. TJs are required to restrict the nonselective passage of small molecules between cells once contacts have been formed. TJs connect cells firmly to each other by OCLN and members of the claudin family of transmembrane proteins [
19]. TJs are linked to the actin cytoskeleton through complexes containing various intracellular proteins such as ZO-1, ZO-2, and ZO-3 [
20]. AJs are composed of a transmembrane protein, E-cad, which is linked indirectly to actin through several proteins, including β-catenin [
21]. TJ formation is closely related to epithelial cell polarization and requires AJ formation [
16].
TJ structures in the airway of asthmatic patients are disrupted [
22]. Furthermore, primary cultured airway epithelial cells obtained from asthmatic patients exhibit immature barrier function in differentiation in vitro and are more prone to damage from cigarette smoke than are healthy individuals [
23]. Epithelial barrier development in asthmatic patients may be impaired by genetic factors, virus infection, inhaled allergens, or air pollution [
22,
24].
PCDH1 co-localized with E-cad at the apical surface of the epithelial cell monolayer, and
PCDH1 knockdown reduced both TJ formation and AJ formation in intercellular spaces at the apical surface in immortalized normal human airway epithelial cell lines (Fig.
7 and Fig.
3b). This suggests that PCDH1 facilitates E-cad assembly, and its loss would inhibit TJ formation through direct or indirect mechanism. Consistent with this, proliferation, apoptosis, or E-cad total levels were not affected by
PCDH1 knockdown (Fig.
5). A limitation of our study is that we did not study the effect of
PCDH1 knockdown on primary cultured epithelial cells because of technical difficulties in achieving sustained siRNA delivery into primary cells.
Local administration of glucocorticoids is the most effective current therapy for bronchial asthma. Recently, we reported that glucocorticoids ameliorated the conditions associated with impaired airway epithelial cell barrier function. Here, we found that the addition of Dex strongly induced expression of PCDH1 isoform-2. This isoform has a long cytoplasmic region, which suggests that it possesses outside-in-signal transduction functionality. Conversely, isoform 1 has no long cytoplasmic region and only has an extracellular region, which suggests that its function differs from that of isoform 2. We also found that the increase in epithelial barrier function was accompanied by an increase in the expression of isoform 2 relative to that of isoform 1. Together, these data suggest that PCDH1 isoform 2 has a positive effect on epithelial barrier formation and that the integrity of barrier function and elevated PCDH1 expression caused by glucocorticoids are probably functionally related events. We will focus on the differences between the functions of individual PCDH1 variants in future studies.
To clarify the contribution of PCDH1 to the pathogenesis of bronchial asthma and CRS, we analyzed the distribution of PCDH1 expression in the airways and nasal mucosal tissue obtained from patients with bronchial asthma and CRS. Human nasal and airway mucosal epithelium mainly is composed of basal cells and CECs. The histological appearance of these epithelia is similar. Here, PCDH1 was mainly expressed in CECs in the airway or nasal epithelium. Considering that the biological barrier function of the epithelium is especially important in the airway, which is constantly in contact with foreign substances, it is not surprising that PCDH1 is highly expressed in the mucosal epithelium of these tissues. We could not compare the expression levels of PCDH1 and other TJs/AJs proteins. But, interestingly, in both asthma and CRS patients, low PCDH1 expression levels in the airway epithelium were observed in regions containing inflammatory cells, large-scale epithelial detachment, and widened intercellular spaces. This suggests that the low PCDH1 expression in this area is associated with increased damage and vulnerability of the epithelial barrier function. We used several sources of lung sections, including autopsy samples obtained from fatal asthmatic patients. All patients were relatively severe asthmatic patients with airflow limitation. In future studies, it will be important to study the expression and function of PCDH1 in larger numbers of subjects with varying asthma severity.
It is likely that PCDH1, which has been identified as an airway hyperreactivity-susceptible gene, plays an important role in epithelial barrier function. In addition, the results of this study make it clear that expression of this type of gene can be induced by glucocorticoids, which are the most effective agents for bronchial asthma. These results improve our understanding of the relationship between epithelial barrier function and allergic airway inflammation. We have demonstrated that the regulation of epithelial barrier function is mediated through PCDH1 and shown that PCDH1 is downregulated in allergic inflammation. Together, these results suggest that restoration of PCDH1 levels and/or function should be a potential therapeutic strategy for the treatment of bronchial asthma or CRS. Thus, the results of our study may contribute to the development of new treatment methods for allergic airway inflammation.
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Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
KY and YG contributed equally as co-first authors. YK, YG, SM, KK, and AS performed in vitro cell experiments, data analysis, drafted the paper, and approved its final version. YK, YG, HK, YN, MI, and SH contributed to the study design and clinical study, drafted the paper, and approved its final version.