Figure 2 | Scientific Reports

Figure 2

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

Figure 2

Hepsin is an activator of proMMP-1 and proMMP-3. (A) Bovine nasal cartilage explants were stimulated with IL-1 (1 ng/ml) and OSM (10 ng/ml) in the presence and absence of matriptase or hepsin (100 nM). Media were removed and the cartilage re-stimulated at day 7. The experiment was terminated as described in the experimental procedures, and the percentage collagen release determined by hydroxyproline assay. Left panel: Data are presented as day 7 collagen release and total collagen release (day 7 + 10). Right panel: Collagenase activity in Day 7 media was monitored by a 3H collagen bioassay. Data are plotted as mean ± SD (n = 6) and representative of 3 independent experiments. Statistical significance was measured by one-way ANOVA with Bonferroni post-hoc test where ***p < 0.001 vs IL-1+OSM. (B) Recombinant full-length proMMP-1 or proMMP-3 were incubated with recombinant hepsin in a 1:5 serine proteinase:MMP molar ratio at 37 °C for up to 24 h. Left panel: Products were separated by 10% SDS-PAGE and gels stained with silver. Right panel: For activity assays, proMMP-1 or proMMP-3 were incubated with hepsin or matriptase for 4 h at 37 °C. Enzyme to substrate ratios were 1:5 for proMMP-1 and 1:10 for proMMP-3. MMP activity was assessed with the MMP-specific substrate FS-6. ProMMP alone or in the presence of APMA were used as negative and positive controls, respectively. Hepsin and matriptase without MMP did not cleave the substrate significantly. Hepsin and matriptase were also pre-treated with the irreversible serine proteinase inhibitor di-isopropyl fluorophosphate (DFP), to demonstrate the involvement of serine proteinase activity in MMP activation. Data are presented as a percentage of APMA-activated proMMP (mean ± SD) following subtraction of the blank measurement. Statistical significance was measured by a one-way ANOVA with Bonferroni post-hoc test where ***p < 0.001 compared to proMMP alone. (C) Day 7 culture media from stimulated bovine cartilage in A were subjected to gelatin zymography. Where gel images have been cropped for clarity, full length images are presented in supplementary information.