Fig. 8

Matriptase zymogen is an efficient PAR-2 activator in the presence of membrane-anchored prostasin and HAI-1. a KOLF cells were transfected with plasmids encoding PAR-2-AP fusion protein and HAI-1 in combination with expression vectors for wildtype (lanes 2, 6, and 9) prostasin, catalytically inactive (S238A, lanes 3, 7, and 10) prostasin, wildtype (lane 4), zymogen-locked matriptase (lanes 5–7), or catalytically inactive matriptase (lanes 8–10). The corresponding empty vectors (EV) for prostasin (lanes 1, 4, 5, and 8) and matriptase (lanes 1–3) were used to ensure that transfections were performed with the same total amount of DNA. Data are shown as mean ± SD of duplicate transfections of free AP released to the media after 4 h standardized to the AP remaining on the cells. **P < 0.0001, one-way ANOVA. The data represent four independent experiments. b HEK293 cells were co-transfected with pSRE-firefly luciferase and pRL-Renilla luciferase reporter plasmids in combination with expression vectors for PAR-2 and HAI-1 (bars 1–12), wildtype (Mat wt) (bars 1–3), zymogen-locked (MatR614Q) matriptase (bars 4–6), catalytically inactive (MatS805A) matriptase (bars 7–9), or corresponding EV (bars 10–12) in combination with wildtype (WT) prostasin (bars 2, 5, 8, and 11), catalytically inactive (S238A) prostasin (bars 3, 6, 9, and 12), or corresponding EV (bars 1, 4, 7, and 10). Data are shown as mean ± SD of the mean of triplicate transfections of firefly luciferase light units/Renilla luciferase light units. **P < 0.0001, one-way ANOVA analysis. The data are representative of three similar experiments. c Zymogen-locked matriptase activity is dependent on membrane anchorage of prostasin. HEK293 cells were co-transfected with pSRE-firefly luciferase and pRL-Renilla luciferase reporter plasmids in combination with expression vectors for PAR-2 (bars 1–8), wildtype (Mat wt) matriptase (bars 1 and 2), zymogen-locked (MatR614Q) (bar 3 and 4) matriptase, catalytically inactive (MatS805A) matriptase (bars 5 and 6), or corresponding EV (bars 7 and 8) and human recombinant prostasin (bars 2, 4, 6, and 8) or vehicle (bars 1, 3, 5, and 7). Data are shown as the mean ± SD of triplicate transfections of firefly luciferase light units/Renilla luciferase light units. *P < 0.0001 and n.s. = not significant, one-way ANOVA. The data are representative of three similar experiments