Abstract
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 Arg36-Ser37, 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 (Ser37-Leu38). 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.
Highlights
The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices
After confirming the peptide was cleaved by known PAR2-cleaving proteinases (Fig. 1B), the peptide was incubated with APMA-activated MMP-1, MMP-8, and MMP-13
We demonstrated that the collagenases MMP-1, -8, and -13 can cleave the PAR2 extracellular region, with MMP-1 yielding a single product resulting from cleavage at Ser37-Leu38 with an additional cleavage at
Summary
The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. We demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKVNH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses. The cleavage of triple-helical collagens such as type II collagen (the major structural component present in cartilage) by the soluble collagenase subset of matrix metalloproteinases (MMP-1, MMP-8, and MMP-13) represents an example of highly specific proteolysis whereby only a single peptide bond is cleavable under normal physiological conditions [3] This initial cleavage leads to collagen denaturation, concomitant with increased susceptibility to other less specific proteinases and disassembly of the collagen network, which is a requirement as cartilage is remodeled into bone during development, and occurs during cartilage breakdown in degenerative scenarios such as rheumatoid arthritis and osteoarthritis (OA). Collagenase cleavage resulted in antagonism of PAR2 activation, suggesting an important negative feedback mechanism whereby canonical PAR2 activation induces MMP expression, and MMP activity can subsequently antagonize PAR2
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