A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation.
BACKGROUND
Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC.


RESULTS
Western blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 microM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37 degrees C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity.


CONCLUSIONS
These data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response.
Alshurafa
Hashem N
HN
Department of Medicine, University of Alberta, Edmonton, AB, Canada, T6G 2S2. hashem@thrombosis.hhscr.org
Stenton
Grant R
GR
Wallace
John L
JL
Hollenberg
Morley D
MD
Befus
A Dean
AD
Vliagoftis
Harissios
H
D000818:Animals; D043422:Carboxypeptidases A / Q000378:metabolism; D053818:Chymases; D066298:In Vitro Techniques; D007249:Inflammation / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D044464:Receptor, PAR-2 / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
C487404:2-furoyl-LIGRLO-amide; D009842:Oligopeptides; D044464:Receptor, PAR-2; D014409:Tumor Necrosis Factor-alpha; D010447:Peptide Hydrolases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; D053818:Chymases