title: Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.
abstract: BACKGROUND
  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 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.
  
  
  METHODS
  To 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.
  
  
  RESULTS
  PE 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.
  
  
  CONCLUSIONS
  PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nomura
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Obata
authors.forename: Takashi
authors.initials: T
authors.lastname: Keira
authors.forename: Ryo
authors.initials: R
authors.lastname: Miyata
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hirakawa
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Takano
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kohno
authors.forename: Norimasa
authors.initials: N
authors.lastname: Sawada
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Himi
authors.affiliation: Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. ktakashi@sapmed.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kojima
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D008666:Metalloendopeptidases / Q000502:physiology*; D009297:Nasal Mucosa / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology*; D010196:Pancreatic Elastase / Q000502:physiology*; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D019108:Tight Junctions / Q000201:enzymology* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D044464:Receptor, PAR-2; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa