Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover
Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover
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DOI：
10.1074/jbc.ra119.006974
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发表时间：
2019-06-28
影响因子：
4.8
通讯作者：
Wilkinson, David J.
中科院分区：
文献类型：
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作者：
Falconer, Adrian M. D.;Chan, Chun Ming;Wilkinson, David J.
基质金属蛋白酶 (MMP) 的胶原酶亚家族在胶原基质的重塑中具有重要作用。蛋白酶激活受体 (PAR) 家族具有独特的激活机制，需要对细胞外结构域进行蛋白水解，形成新 N 末端，充当束缚配体，这一过程与关节炎的发展相关。典型的 PAR2 激活通常通过 Arg(36)-Ser(37) 处的丝氨酸蛋白酶发生，但其他蛋白酶可以裂解束缚配体下游的 PAR 并解除受体的武装。为了识别 PAR2 内的其他切割位点，我们合成了与胞外区域相对应的 42 个氨基酸肽。我们观察到所有三种可溶性 MMP 胶原酶 MMP-1、MMP-8 和 MMP-13 均裂解 PAR2 并发现了一个新的裂解位点（Ser(37)-Leu(38)）。来自再吸收牛鼻软骨的金属蛋白酶和重组人胶原酶可以切割模拟典型 PAR2 激活区域的猝灭荧光肽，并测定了动力学常数。在 PAR2 过表达的 SW1353 软骨细胞中，我们证明激活肽 SLIGKV-NH2 诱导快速钙流、炎症基因表达（包括 MMP1 和 MMP13）以及细胞外信号调节激酶 (ERK) 和 p38 激酶的磷酸化。相应的 MMP 裂解衍生肽 (LIGKVD-NH2) 没有表现出典型的激活作用；然而，我们观察到 ERK 的磷酸化，提供了偏向激动的证据。重要的是，我们证明，与活性 MMP-1 预孵育可减少经典激活剂 matriptase 对下游 PAR2 的激活，但不会减少 SLIGKV-NH2 的激活。这些结果支持胶原酶作为能够解除 PAR2 的蛋白酶的作用，揭示了抑制 PAR2 介导的炎症反应的机制。
The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg(36)-Ser(37), but other proteinases can cleave PARs downstream of the tethered ligand and disarm the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser(37)-Leu(38)). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2. These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.