UBC James Hogg Research Centre, Institute for Heart+Lung Health, St. Paul's Hospital, Vancouver, British Columbia, Canada.
PLoS One. 2012;7(3):e33163. doi: 10.1371/journal.pone.0033163. Epub 2012 Mar 30.
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.
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.
颗粒酶 B（GrB）是一种促凋亡的丝氨酸蛋白酶，有助于免疫介导的靶细胞凋亡。然而，在炎症过程中，GrB 会积聚在细胞外空间，保持其活性，并能够切割细胞外基质（ECM）蛋白。最近的研究表明，GrB 在心血管疾病中具有致病的细胞外作用，但细胞外 GrB 活性的病理生理后果在很大程度上仍不清楚。本研究的目的是鉴定 GrB 的蛋白聚糖（PG）底物，并研究 GrB 释放 PG 结合的 TGF-β1 进入细胞外环境的能力。
方法/结果：鉴定出三种细胞外 GrB PG 底物；decorin、biglycan 和 betaglycan。由于所有这些 PG 都能结合活性 TGF-β1，因此进行了细胞因子释放测定，以确定 GrB 介导的 PG 切割是否诱导 TGF-β1 释放。我们的数据证实 GrB 从所有三种底物以及内源性 ECM 中释放 TGF-β1，而 GrB 抑制剂 3,4-二氯异香豆素抑制了这一过程。释放的 TGF-β1 保留其活性，这可通过诱导人冠状动脉平滑肌细胞中 SMAD-3 磷酸化来证明。
除了有助于 ECM 降解和体内组织结构完整性丧失外，增加的细胞外 GrB 活性还能够诱导 PG 中活性 TGF-β1 的释放。
