Pro-Inflammatory Actions of the Exodomain Shed From Protease Activated Receptor 2 (PAR-2)

Abstract

Rationale: Activation of PAR-2 by proteases has been implicated as a key mechanism in allergic and other inflammatory conditions. Proteolytic cleavage can lead to exposure of a tethered ligand whose binding to the receptor stimulates cell signalling, and the release from PAR-2 of an exodomain fragment (PAR-2E). Potential functions of PAR-2E have been little studied. Methods: PAR-2E was synthesised and its stability investigated in human biological fluids, cell supernatants and with selected proteases. The ability of PAR-2E to stimulate calcium flux in cultures of human umbilical vein endothelial cells was examined using a fluorescence based microplate procedure, and altered RNA expression by whole genome microarray analysis and quantitative PCR (qPCR) for selected genes. Adhesion molecule expression was investigated by flow cytometry. In parallel studies, nucleated cells were enumerated and levels of matrix metalloproteases (MMP) determined by gelatin zymography in peritoneal lavage fluid from C57BL/6 mice injected intraperitoneally with PAR-2E. Results: PAR-2E was highly susceptible to degradation by proteases, but its addition to cells resulted in calcium flux, and increased expression of genes for various adhesion molecules (including ICAM1, EPCAM and ITGAL), and cytokines (TNFAIP3 and IL1B). PAR-2E-induced upregulation of ICAM1, VCAM-1, EPCAM and ITGAL was observed on flow cytometry. Injection of PAR-2E into mice was associated with eosinophilia and raised levels of MMP2 in peritoneal lavage fluid. Conclusions: PAR-2E may act as a stimulus for increased expression of adhesion molecules, cytokine release and eosinophilia, and deserves consideration as a mediator of inflammation following PAR-2 activation.