Figure 5 | Scientific Reports

Figure 5

From: The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Figure 5

Hepsin is a weak PAR2 activator. (A) To assess the ability of hepsin to activate PAR2 in vivo, hepsin (5 µg) was administered topically to an exposed knee joint of both PAR2+/+ (; n = 3) and PAR2−/− (; n = 4) mice, with synovial perfusion monitored by high resolution Doppler imaging, as described previously5. Data are presented as percentage change in perfusion from the baseline mean ± SEM. (B) Hepsin or matriptase (10–100 nM) were added to SW1353 cells stably expressing PAR2 for 3 hours. Cells were lysed, mRNA reverse transcribed and IL8 expression monitored by real-time PCR. Data were normalized to an 18S rRNA housekeeping gene and plotted as fold change compared to unstimulated cells. (C) Matriptase is a more potent PAR2 activator. Left panel: Hepsin (1 nM) or matriptase (1 nM) were incubated with 2-Abz-SKGRSLIG-Y(NO2) (0–200 μM), fluorescence monitored and linear reaction velocities obtained from progress curves. Velocities were plotted against substrate concentration and Michaelis-Menten constants generated by non-linear regression. Right panel: Expanded view of the non-linear regression for hepsin confirming normal Michaelis-Menten kinetics. Curves are representative of 3 independent experiments. Tabulated kinetic constants are also shown as mean ± SD across each of 3 independent experiments.