Figure 1.
GrB-mediated cleavage of decorin, biglycan and betaglycan.
Increasing concentrations of GrB (25, 50, 100 and 200 nM) were incubated with decorin (a), biglycan (b), and betaglycan (c) for 24 h at RT. * denotes full length protein, arrows indicate cleavage fragments and ∧ indicates GrB.
Figure 2.
GrB-mediated PG cleavage is inhibited by DCI and cleavage site identification.
GrB was incubated with decorin (a), biglycan (b) and betaglycan (c), +/− DCI and the solvent control DMSO, for 4 h and 24 h. Cleavage sites in biglycan and betaglycan were identified by N-terminal Edman degradation. * denotes full length protein, arrows indicate cleavage fragments, and cleavage sites are displayed on the right.
Figure 3.
GrB cleaves native smooth muscle cell-derived decorin and biglycan.
HCASMCs were incubated at confluency for adequate ECM synthesis. Cells were removed, GrB was incubated with the ECM and decorin and biglycan cleavage fragments were detected by western immunoblotting.
Figure 4.
GrB-mediated cleavage of decorin, biglycan and betaglycan results in the release of active TGF-β1.
Decorin, biglycan and betaglycan complexed with TGF-β1 were treated with GrB. Supernatants (containing released TGF-β1), were collected and released TGF-β1 was detected by Western blotting. Results shown are representative western blots from at least 3 separate experiments for each PG (a). As endogenous SMC-derived ECM only contains latent TGF-β (as shown in (b)), GrB-mediated release from active TGF-β1 supplemented ECM was also examined (c).
Figure 5.
TGF-β1 released by GrB is active and induces SMAD-3 activation in HCASMCs.
GrB+/−DCI was incubated on betaglycan/TGF-β1 complexes for 24 h. Supernatants (containing released TGF-β1) were added to HCASMC for 20 m and phosphorylated SMAD-2 and SMAD-3 levels were examined. TGF-β1 released by GrB is active and induces SMAD-3 signalling in HCASMCs (P<0.05). The result shown is representative of at least 5 experiments.