Granzyme B Cleaves Decorin, Biglycan and Soluble Betaglycan, Releasing Active Transforming Growth Factor-β1

Objective: Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-b1 into the extracellular milieu. Methods/Results: Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-b1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-b1 release. Our data confirmed that GrB liberated TGF-b1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-b1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. Conclusion: In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increase

GrB-mediated PG cleavage is inhibited by DCI and cleavage site identification.

<p>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.</p

GrB-mediated cleavage of decorin, biglycan and betaglycan.

<p>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.</p

TGF-β1 released by GrB is active and induces SMAD-3 activation in HCASMCs.

<p>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.</p