Abstract
BackgroundPseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown.MethodsTo investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function.ResultsPE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs.ConclusionsPE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.
Highlights
Pseudomonas aeruginosa (P. aeruginosa) is a virulent Gram-negative bacterium that causes aggressive infections in patients compromised by pre-existing respiratory disease such as cystic fibrosis and diffuse panbronchiolitis [1,2]
P. aeruginosa elastase (PE) transiently reduces the expression of transmembrane proteins in the tight junctions in human nasal epithelial cells (HNECs) To investigate whether Pseudomonas aeruginosa elastase (PE) affects the protein and mRNAs expression of tight junction and adherens junction molecules in HNECs, hTERT-HNECs were treated with 0.1 U PE for 30 min, 1 h, 2 h, and 4 h
Western blots showed that claudin-1, -4, and tricellulin protein levels decreased at 30 min but were restored at 2 h, whereas occludin protein was transiently reduced at 1 h (Figure 1)
Summary
Pseudomonas aeruginosa (P. aeruginosa) is a virulent Gram-negative bacterium that causes aggressive infections in patients compromised by pre-existing respiratory disease such as cystic fibrosis and diffuse panbronchiolitis [1,2]. P. aeruginosa is associated with prolonged chronic rhinosinusitis (CRS) [3]. P. aeruginosa elastase (PE) increases paracellular permeability in lung epithelial cells via mechanisms involving tight junction disruption and cytoskeletal reorganization [6]. PKC signaling is involved in PE-induced epithelial barrier disruption via tight junction translocation and cytoskeletal reorganization in the human bronchial adenocarcinoma cell line Calu-3 [2]. Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is implicated in prolonged chronic rhinosinusitis. The effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown
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