Limits...
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.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316562&req=5

pone-0033163-g004: 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).

Mentions: As decorin, biglycan and betaglycan sequester active TGF-β1 [22], [23], TGF-β1 release assays were performed to determine if GrB-mediated cleavage of these proteins resulted in active TGF-β1 release (Fig. 4a). Following 24 h of incubation, minimal TGF-β1 had dissociated from the plate in the absence of GrB, suggesting that the PG/TGF-β1 complexes were stable throughout the incubation time. After 24 h of GrB treatment, TGF-β1 was released into the supernatants, from all three PGs. This release was inhibited by DCI, suggesting the process was dependent on active GrB. TGF-β1 release was observed at GrB levels as low as 25 nM (Unpublished observations).


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033163-g004: 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).
Mentions: As decorin, biglycan and betaglycan sequester active TGF-β1 [22], [23], TGF-β1 release assays were performed to determine if GrB-mediated cleavage of these proteins resulted in active TGF-β1 release (Fig. 4a). Following 24 h of incubation, minimal TGF-β1 had dissociated from the plate in the absence of GrB, suggesting that the PG/TGF-β1 complexes were stable throughout the incubation time. After 24 h of GrB treatment, TGF-β1 was released into the supernatants, from all three PGs. This release was inhibited by DCI, suggesting the process was dependent on active GrB. TGF-β1 release was observed at GrB levels as low as 25 nM (Unpublished observations).

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