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.

Mast cells and mast cell tryptase enhance migration of human lung fibroblasts through protease-activated receptor 2

“…This leads to the activation of downstream cell signaling pathways, involving binding to β-arrestins and activating ERK1, 2 [ 16 ]. PAR2 is expressed by several cell types and may be involved in tissue remodeling by inducing fibroblast migration, differentiation and ECM production [ 17 , 18 ]. The expression of PAR2 has been reported to be elevated in fibroblasts and myofibroblasts in IPF [ 19 ], as well as in inflammatory diseases [ 20 ].…”

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

“…Bleomycin-induced pulmonary fibrosis is stably formed at around d 14, which is consequently frequently used as an endpoint in the literature (19,28,37). However, the fibrotic stage induced by bleomycin peaks at around 3-4 wks and the reduction in fibrosis observed in PAR-2-deficient mice at d 14 (19,30) may thus indicate only a delay in the onset of fibrosis instead of reduced progression. To address this concern, we increased the follow-up time and showed that the severity of fibrosis as well as collagen accumulation are still significantly reduced in PAR-2-deficient mice compared with wild-type mice 28 d after bleomycin instillation.…”

“…We confirm and extend these findings by showing that P2pal-18S effectively blocks PAR-2-driven profibrotic responses in vitro. Indeed, preincubation of murine fibroblasts with P2pal-18S blocks trypsin-induced extracellular signal-regulated kinases (ERK) phosphorylation, reduces proliferation and mi- gration (30) and diminishes α-SMA and collagen expression (Figure 2). Moreover, P2pal-18S significantly inhibits collagen expression not only in control fibroblasts but also in IPF-derived human fibroblasts.…”

“…We previously showed that PAR-2 deficiency limits bleomycin-induced pulmonary fibrosis when we analyzed mice 14 d after bleomycin instillation (19,30). To determine whether PAR-2 indeed drives the progression of pulmonary fibrosis or whether its absence would merely delay the onset of fibrosis, we compared lungs from wild-type and PAR-2-deficient mice at d 28 and 48 after the instillation of bleomycin.…”

Targeting coagulation factor receptors – protease‐activated receptors in idiopathic pulmonary fibrosis

“…PAR-1 also promotes profibrotic effects by facilitating TGF-b activation, inducing the differentiation of fibroblasts into myofibroblasts, and stimulating ECM synthesis [43,75]. In addition, PAR-1 seems to synergize with PAR-3 to mediate EMT of alveolar epithelial cells [76], and with PAR-2 to induce fibrotic effects on fibroblasts [77]. The importance of these in vitro findings is emphasized by the fact that genetic ablation of PAR-1 limits bleomycin-induced acute lung inflammation and fibrosis, as is evident from reduced total collagen levels in the lung in combination with decreased levels of proinflammatory and profibrotic mediators, such as TGF-b, IL-6, and MCP-1 [78].…”