The online version of this article (doi:10.1186/1465-9921-15-21) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interest.
KN and TK carried out the genetic cell biological studies and drafted the manuscript. KO, RM and SH participated in cell culture. KT participated in the design of the study and performed the statistical analysis. TH and SN conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
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.
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.
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.
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.
Additional file 1: (A) Western blotting for tight junction and adherens junction proteins in hTERT-transfected HNECs after treatment with 0.01 U neutrophil elastase. (B) RT-PCR for mRNAs of tight junction molecules in hTERT-transfected HNECs after treatment with 0.01 U neutrophil elastase. NE: neutrophil elastase. (PDF 185 KB)
Additional file 2: Western blotting for tight junction proteins in hTERT-transfected HNECs treatment with pan-PKC inhibitor (GF109203X), MEK1/2 inhibitor (U0126), PI3K inhibitor (LY294002), p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), epidermal growth factor (EGF) receptor inhibitor (AG1478), COX1 inhibitor (FR122047), and COX2 inhibitor, NF-κB inhibitor (IMD-0354), and Proteasome inhibitor (MG132) without Pseudomonas aeruginosa elastase.(PDF 141 KB)
Leduc D, Beaufort N, de Bentzmann S, Rousselle JC, Namane A, Chignard M, Pidard D: The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis. Infect Immun. 2007, 75: 3848-3858. 10.1128/IAI.00015-07. PubMedPubMedCentralCrossRef
Cosgrove S, Chotirmall SH, Greene CM, McElvaney NG: Pulmonary proteases in the cystic fibrosis lung induce interleukin 8 expression from bronchial epithelial cells via a heme/meprin/epidermal growth factor receptor/Toll-like receptor pathway. J Biol Chem. 2011, 286: 7692-7704. 10.1074/jbc.M110.183863. PubMedPubMedCentralCrossRef
Yeoh S, Church M, Lackie P, McGill J, Mota M, Hossain P: Increased conjunctival expression of protease activated receptor 2 (PAR-2) in seasonal allergic conjunctivitis: a role for abnormal conjunctival epithelial permeability in disease pathogenesis?. Br J Ophthalmol. 2011, 95: 1304-1308. 10.1136/bjo.2010.191221. PubMedCrossRef
Sawada N, Murata M, Kikuchi K, Osanai M, Tobioka H, Kojima T, Chiba H: Tight junctions and human diseases. Med Elecron Microsc. 2003, 36: 147-156. 10.1007/s00795-003-0219-y. CrossRef
Schneeberger EE, Lynch RD: The tight junction: a multifunctional complex. Am J Physiol Cell Physiol. 2004, 286: 1213-1228. 10.1152/ajpcell.00558.2003. CrossRef
Kojima T, Murata M, Yamamoto T, Lan M, Imamura M, Son S, Takano K, Yamaguchi H, Ito T, Tanaka S, Chiba H, Hirata K, Sawada N: Tight junction proteins and signal transduction pathways in hepatocytes. Histol Histopathol. 2009, 24: 1463-1472. PubMed
Kojima T, Go M, Takano K, Kurose M, Ohkuni T, Koizumi J, Kamekura R, Ogasawara N, Masaki T, Fuchimoto J, Obata K, Hirakawa S, Nomura K, Keira T, Miyata R, Fujii N, Tsutsumi H, Himi T, Sawada N: Regulation of tight junctions in upper airway epithelium. Biomed Res Int. 2013, 2013: 947072- PubMedPubMedCentral
Kurose M, Kojima T, Koizumi JI, Kamekura R, Ninomiya T, Murata M, Ichimiya S, Osanai M, Chiba H, Himi T, Sawada N: Induction of claudins in passaged hTERT-transfected human nasal epithelial cells with an extended life span. Cell Tissue Res. 2007, 330: 63-74. 10.1007/s00441-007-0453-z. PubMedCrossRef
Koizumi J, Kojima T, Ogasawara N, Kamekura R, Kurose M, Go M, Harimaya A, Murata M, Osanai M, Chiba H, Himi T, Sawada N: Protein kinase C enhances tight junction barrier function of human nasal epithelial cells in primary culture by transcriptional regulation. Mol Pharmacol. 2008, 74: 432-442. 10.1124/mol.107.043711. PubMedCrossRef
Ohkuni T, Kojima T, Ogasawara N, Masaki T, Fuchimoto J, Kamekura R, Koizumi JI, Ichimiya S, Murata M, Tanaka S, Himi T, Sawada N: Poly(I:C) reduces expression of JAM-A and induces secretion of IL-8 and TNF-α via distinct NF-κB pathways in human nasal epithelial cells. Toxicol Appl Pharmacol. 2011, 250: 29-38. 10.1016/j.taap.2010.09.023. PubMedCrossRef
Obata K, Kojima T, Masaki T, Okabayashi T, Yokota S, Hirakawa S, Nomura K, Takasawa A, Murata M, Tanaka S, Fuchimoto J, Fujii N, Tsutsumi H, Himi T, Sawada N: Curcumin prevents replication of respiratory syncytial virus and the epithelial responses to it in human nasal epithelial cells. PLoS One. 2013, 8: e70225-10.1371/journal.pone.0070225. PubMedPubMedCentralCrossRef
- Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells
- BioMed Central
Neu im Fachgebiet Innere Medizin
Meistgelesene Bücher aus der Inneren Medizin
Mail Icon II