Human airway trypsin-like protease stimulates differentiation of fibroblasts to myofibroblasts in human bronchial fibroblasts in vitro

Background and Methods: Human airway trypsin-like protease (HAT) is a monomeric serine protease isolated from the mucoid sputum of patients with chronic airway diseases like bronchial asthma. To examine whether HAT can induce differentiation of human bronchial fibroblasts (HBFs) into myofibroblasts, the effects of HAT on expression of α-smooth muscle actin (αSMA) in HBFs cultured from human bronchial tissues were investigated with Western blot and immunofluorescence. Results: Immunofluorescence showed that HAT increased cell size and significantly increased intracellular αSMA fiber expression in HBFs. HAT at 25-200 mU/ml significantly increased αSMA expression on Western blot analysis. Both human thrombin at 5 nM and protease activated receptor (PAR) 1 activating peptide (SRLLRN) at 400 µM enhanced αSMA expression to a similar extent to HAT at 100 mU/ml. RT PCR of PAR1, 2, 3, and 4 in HBFs showed that the expression of the mRNA of PAR1 was much more marked than the expressions of the other PARs. Immunofluorescence staining of PAR1 showed that thrombin and SRLLRN caused internalization of PAR1 at the cell surface, but HAT did not. With respect to the effect of HAT on the N-terminal peptide of PAR1, 37TLDPRSFLLRNPNDK51, HAT cleaved both the R41-S42 bond (acivating side) and the R46-N47 bond (the noncanonical activating site) of PAR. The stimulatory effect of HAT on αSMA expression was significantly diminished by knockdown of PAR1. Conclusions: These results indicate that HAT stimulates differentiation of HBFs via PAR1, and that HAT is related to repair of tissue injury in the airway.