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Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-β1.

Boivin WA, Shackleford M, Vanden Hoek A, Zhao H, Hackett TL, Knight DA, Granville DJ - PLoS ONE (2012)

Bottom Line: Our data confirmed that GrB liberated TGF-β1 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-β1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells.In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for Heart+Lung Health, St. Paul's Hospital, Vancouver, British Columbia, Canada.

ABSTRACT

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-β1 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-β1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-β1 release. Our data confirmed that GrB liberated TGF-β1 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-β1 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, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.

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Related in: MedlinePlus

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.
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pone-0033163-g005: 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.

Mentions: To determine that the TGF-β1 released by GrB remained active and was not bound to an inhibitory fragment, supernatants from the betaglycan release assay were incubated on HCASMC for 20 min (Fig. 5). TGF-β signaling was examined through the phosphorylation and activation of SMAD-3. HCASMC responded well to 5 ng/ml TGF-β1, with SMAD-3 phosphorylation observed at 20 min (P<0.05). The TGF-β1 released from betaglycan by GrB induced SMAD-3 signaling, confirming that it remained active (p<0.05). Total SMAD-3 levels also did not change.


Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-β1.

Boivin WA, Shackleford M, Vanden Hoek A, Zhao H, Hackett TL, Knight DA, Granville DJ - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316562&req=5

pone-0033163-g005: 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.
Mentions: To determine that the TGF-β1 released by GrB remained active and was not bound to an inhibitory fragment, supernatants from the betaglycan release assay were incubated on HCASMC for 20 min (Fig. 5). TGF-β signaling was examined through the phosphorylation and activation of SMAD-3. HCASMC responded well to 5 ng/ml TGF-β1, with SMAD-3 phosphorylation observed at 20 min (P<0.05). The TGF-β1 released from betaglycan by GrB induced SMAD-3 signaling, confirming that it remained active (p<0.05). Total SMAD-3 levels also did not change.

Bottom Line: Our data confirmed that GrB liberated TGF-β1 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-β1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells.In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for Heart+Lung Health, St. Paul's Hospital, Vancouver, British Columbia, Canada.

ABSTRACT

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-β1 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-β1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-β1 release. Our data confirmed that GrB liberated TGF-β1 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-β1 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, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.

Show MeSH
Related in: MedlinePlus