Protease‐activated receptor ( PAR )‐2 is required for PAR ‐1 signalling in pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease‐activated receptor ( PAR )‐1 and PAR ‐2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin‐induced lung fibrosis is diminished in both PAR ‐1 and PAR ‐2 deficient mice. We thus have been suggested that combined inactivation of PAR ‐1 and PAR ‐2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR ‐1 and PAR ‐2 agonists in the absence or presence of specific PAR ‐1 or PAR ‐2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild‐type and PAR ‐2 deficient mice with or without a specific PAR ‐1 antagonist (P1pal‐12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR ‐1 and/or PAR ‐2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR ‐1 and PAR ‐2 did not show any additive effects on these pro‐fibrotic responses. Strikingly, PAR ‐2 deficiency as well as pharmacological PAR ‐1 inhibition reduced bleomycin‐induced pulmonary fibrosis to a similar extent. PAR ‐1 inhibition in PAR ‐2 deficient mice did not further diminish bleomycin‐induced pulmonary fibrosis. Finally, we show that the PAR ‐1‐dependent pro‐fibrotic responses are inhibited by the PAR ‐2 specific antagonist. Targeting PAR ‐1 and PAR ‐2 simultaneously is not superior to targeting either receptor alone in bleomycin‐induced pulmonary fibrosis. We postulate that the pro‐fibrotic effects of PAR ‐1 require the presence of PAR ‐2.