Protocadherin-1 is a glucocorticoid-responsive critical regulator of airway epithelial barrier function

Yutaka Kozu, Yasuhiro Gon, Shuichiro Maruoka, Kuroda Kazumichi, Akiko Sekiyama, Hiroyuki Kishi, Yasuyuki Nomura, Minoru Ikeda, Shu Hashimoto, Yutaka Kozu, Yasuhiro Gon, Shuichiro Maruoka, Kuroda Kazumichi, Akiko Sekiyama, Hiroyuki Kishi, Yasuyuki Nomura, Minoru Ikeda, Shu Hashimoto

Abstract

Background: Impaired epithelial barrier function renders the airway vulnerable to environmental triggers associated with the pathogenesis of bronchial asthma. We investigated the influence of protocadherin-1 (PCDH1), a susceptibility gene for bronchial hyperresponsiveness, on airway epithelial barrier function.

Methods: We applied transepithelial electric resistance and dextran permeability testing to evaluate the barrier function of cultured airway epithelial cells. We studied PCDH1 function by siRNA-mediated knockdown and analyzed nasal or bronchial tissues from 16 patients with chronic rhinosinusitis (CRS) and nine patients with bronchial asthma for PCDH1 expression.

Results: PCDH1 was upregulated with the development of epithelial barrier function in cultured airway epithelial cells. Immunocytochemical analysis revealed that PCDH localized to cell-cell contact sites and colocalized with E-cadherin at the apical site of airway epithelial cells. PCDH1 gene knockdown disrupted both tight and adhesion junctions. Immunohistochemical analysis revealed strong PCDH1 expression in nasal and bronchial epithelial cells; however, expression decreased in inflamed tissues sampled from patients with CRS or bronchial asthma. Dexamethasone (Dex) increased the barrier function of airway epithelial cells and increased PCDH1 expression. PCDH1 gene knockdown eradicated the effect of Dex on barrier function.

Conclusion: These results suggest that PCDH1 is important for airway function as a physical barrier, and its dysfunction is involved in the pathogenesis of allergic airway inflammation. We also suggest that glucocorticoids promotes epithelial barrier integrity by inducing PCDH1.

Figures

Fig. 1
Fig. 1
Expression of protocadherin-1 (PCDH1) protein in human bronchial epithelial (16HBE) cells. a Time course of PCDH1 protein expression in 16HBE cells. Cell lysates were harvested at the indicated time points. Western blot with anti-PCDH1 antibody showed two bands (150 and 170 kDa) corresponding to PCDH1 isoforms 1 and 2, respectively. β-Actin was used as an internal standard. b Graphs showing densitometric quantification of the PCDH1 isoform 1 or 2 bands on western blots, relative to β-actin. c Immunocytochemical analysis of PCDH1 (red) and cellular nuclei (blue) in 16HBE cells cultured for 24 and 72 h. The data represent the mean of three independent experiments
Fig. 2
Fig. 2
Gene knockdown efficacy of protocadherin-1 (PCDH1)-specific siRNAs. Quantification of PCDH1 mRNA by real-time polymerase chain reaction. a mRNA was purified from the cells harvested at 24 h after transfection of the control (siCtl) or PCDH1-specific siRNAs (siPCDH1_1, siPCDH1_2, and siPCDH1_3). Results are expressed relative to the control value (siCtl-treated cells) and are mean ± SD values; n = 3 independent samples. Asterisks indicate a statistically significant difference (p ≤ 0.05) in the result between that of cells treated with siCtl. b Time course of PCDH1 mRNA expression after PCDH1 siRNA transfection. mRNA was purified from cells harvested at indicated time points after the transfection of siCtl or siPCDH1_1. Results are expressed relative to the control value (siCtl-treated cells at day 1) and are mean ± SD values; n = 3 independent samples. c Time course of PCDH1 protein expression after PCDH1 siRNA transfection. Cell lysates were harvested at the indicated time points after siCtl or siPCDH1_1 transfection and western blotted with anti-PCDH1 antibody. The data represent the mean from three independent experiments (upper photograph). The lower graph shows densitometric quantification of PCDH1 bands on western blots, relative to β-actin. Results are expressed as a relative density compared to the control value (siCtl-treated cells) and are mean ± SD values; n = 3 independent samples. Asterisk indicates a statistically significant difference (p ≤ 0.05) in the result between that of cells treated with siCtl. NS: not significant
Fig. 3
Fig. 3
siRNA knockdown of protocadherin-1 (PCDH1) impairs epithelial barrier formation. Effect of PCDH1 knockdown on barrier development. Human bronchial epithelial (16HBE) cells were transfected with control (siCtl) or PCDH1-specific siRNAs (siPCDH1_1, siPCDH1_2, and siPCDH1_3). After 24 h, cells were seeded onto the Transwell inserts, and transepithelial electrical resistance (a. left) and dextran permeability (a. right) were measured at day 3. Results are expressed as a percentage of the control value (siCtl-treated cells) and are mean ± SD values; n = 3 independent samples. Asterisk indicates a statistically significant difference (p ≤ 0.05) in the result between that of cells treated with siCtl. 1HAE and Calu-3 cells were transfected with control (siCtl) or PCDH1-specific siRNAs (siPCDH1_1). After 24 h, cells were seeded onto the Transwell inserts, and TER (b. left) and dextran permeability (b. right) were measured at day 3. Results are expressed as a percentage of the control value (siCtl-treated cells) and are mean ± SD (n = 3). Asterisks indicate a statistically significant difference (P ≤ 0.05) compared to cells treated with siCtl
Fig. 4
Fig. 4
Silencing of PCDH1 does not affect cell growth or apoptosis. 16HBE cells were transfected with control (siCtl) or PCDH1-specific siRNA (siPCDH1_1). Results are mean ± SD. (n = 3). (left) The number of live cells was counted daily for 3 days using trypan blue. (right) Apoptosis was detected by Annexin V/PI staining at day 3 after transfection. The x-axis shows AnnexinV-FITC binding and the y-axis pertains to the results for staining with the vital dye propidium iodide. Cells in the lower left quadrant are viable, cells in the lower right are apoptotic, and those in the upper right are late stage apoptotic/dead cells
Fig. 5
Fig. 5
Protocadherin-1 (PCDH1) knockdown in airway epithelial cells inhibits the formation of adherence junctions and tight junctions. Immunocytochemical analysis of E-cadherin (E-cad) (a) and zonula occludens-1 (b) in PCDH1-knockdown cells. Human bronchial epithelial (16HBE) cells were transfected with control (siCtl) and PCDH1-specific siRNA (siPCDH1_1). After 48 h, cells were subjected to immunocytochemical analysis with antibodies specific to E-cad (green) or PCDH1 (green). The images represent data from three independent experiments. a Colocalization of PCDH1 and E-cad. 16HBE cells were cultured for 3 days and stained with anti-PCDH1 and anti- E-cad antibodies. Expression of PCDH1 was mainly co-localized with E-cad at the apical site of cell junctions (upper panel); however, the expression level of PCDH1 was relatively low (lower panel). The images represent data from three independent experiments (c)
Fig. 6
Fig. 6
Time course of E-cadherin, ZO-1, and occludin protein expression after PCDH1 knockdown. Cell lysates were harvested at the indicated time points after the transfection of siCtl or siPCDH1_1 and western blotted with anti-PCDH1 antibody. Lower graph shows densitometric quantification of E-cadherin, ZO-1, and occludin bands on western blots, relative to β-actin
Fig. 7
Fig. 7
Role of protocadherin-1 (PCDH1) in dexamethasone (Dex)-mediated epithelial barrier integrity. b Human bronchial epithelial (16HBE) cells were cultured on Transwell inserts with or without Dex. The cell lysates were harvested 72 h after the culture. Western blot with the anti-PCDH1 antibody showed 150- and 170-kDa bands, corresponding to PCDH1 isoforms 1 and 2, respectively. The graph on the right shows the relative expression of PCDH1 isoforms 1 and 2 on western blots quantified by densitometry. The image and graphs represent data from two independent experiments. b PCDH1 knockdown eradicated the enhanced effect of Dex on the development of transepithelial electrical resistance (left) and reduction of dextran permeability (right) in 16HBE cells. Results are expressed as a percentage of the control value (siCtl-treated cells) and are mean ± SD values; n = 3 independent experiments. Asterisks indicate a statistically significant difference (p ≤ 0.05) in the result between that of cells treated with siCtl
Fig. 8
Fig. 8
Protocadherin-1 (PCDH1) expression in the nasal tissues of patients with chronic rhinosinusitis (CRS) and the airway of asthmatic patients. a Representative image of PCDH1 expression in the nasal tissue from a patient with CRS (a,b). PCDH1 expression of normal region (a) and inflamed region (b) in the nasal tissues of CRS. Arrow shows ciliated airway epithelial cells (CECs) in the inflamed region. b. Immunohistochemistry score for PCDH1 expression in CECs of patients with CRS (n = 16). The graph shows the expression level of PCDH1 in the normal region and inflamed region. The horizontal bars represent the mean values of the expression score. An asterisk indicates a statistically significant difference (p ≤ 0.05). c A representative image of PCDH1 expression in the airway of a patient with asthma (a,b). Normal region (a) and inflamed region (b) in the airway tissues of an individual with asthma. Arrow shows asthma in the inflamed region. Arrowhead shows endothelial cells. d Immunohistochemistry score for PCDH1 expression of CECs in asthmatic patients (n = 9). The graph shows the expression levels of PCDH1 in the normal region and inflamed region of the lung tissues. EDC stands for endothelial cells. Horizontal bars represent mean value expression scores. Asterisks indicate a statistically significant difference (p ≤ 0.05)

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