Human airway trypsin-like protease stimulates protease activated receptors and interactions of these receptors

Abstract

Background and Methods: Human airway trypsin-like protease (HAT) was isolated from the mucoid sputum in airway diseases like bronchial asthma and have indicated that HAT achieves its biological effects via activation of protease activated receptor (PAR)2 in several airway cells. In this work, we examined whether HAT can activate PAR receptors in human primary bronchial epithelial cells (HBECs) by transfecting siRNA of PAR1, PAR2 and PAR3 into HBECs, real-time PCR and immunofluorescence. Results: 1) In control siRNA transfected-HBECs, HAT significantly enhanced expression of PAR1, PAR2, PAR3, and IL-8 mRNAs, PAR1 agonist peptide (PAR1 AP) enhanced PAR1, PAR2 and IL-8 mRNAs, and PAR2 AP enhanced PAR2 and IL-8 mRNAs, but did not enhance PAR1 mRNA. 2) In PAR2 siRNA-transfected HBECs, HAT-induced PAR1, PAR2, PAR3 and IL-8 mRNA elevations and PAR2 AP-induced PAR2 and IL-8 mRNA elevations were almost completely suppressed. 3) In the PAR3 siRNA-transfected HBECs, HAT-induced PAR3 and IL-8 mRNAs were completely suppressed, but PAR1 AP-induced PAR1 mRNA elevation and PAR2 AP-induced PAR2 mRNA were not significantly suppressed. 4) In PAR1 siRNA-transfected HBECs, the PAR1-induced PAR1, PAR2 mRNA elevation were completely suppressed, and both HAT-induced PAR2 mRNA and IL-8 mRNA elevation also suppressed. Immunofluorescence methods showed that PAR2 and PAR3 were localized to the cell surface of HBECs and were internalized by stimulation of HAT. Conclusions: These results indicate that HAT may attack and activate PAR1, PAR2 and PAR3 in HBECs. These PARs, particularly PAR2 and PAR3, are intimately associated with IL-8 synthesis in HBECs, by mutual interrelations.