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A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation.

Alshurafa HN, Stenton GR, Wallace JL, Hollenberg MD, Befus AD, Vliagoftis H - BMC Pharmacol. (2004)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Alberta, Edmonton, AB, Canada, T6G 2S2. hashem@thrombosis.hhscr.org

ABSTRACT

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.

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Release of RMCP-1, -5 and CPA from PMC following activation with tc-LIG (PAR-2ap), compound 48/80 and Ag. (A) Supernatants from tc-LIG (10 μM), Ag (10 We/mL) and sham-treated (spon) mast cells were concentrated (10 ×) and Western blot analysis preformed for CPA, RMCP-1 and RMCP-5. Left panel shows Coomassie blue staining of the same gel and right panel Western blot with normal rabbit serum as a negative control. (B) Release of RMCP-1, RMCP-5 and CPA following 20 min and 8 h activation of PMC with tc-LIG (10 μM). (C) Dose response for the release of RMCP-1, -5 and CPA by tc-LIG-stimulated or compound 48/80-stimulated PMC. In all cases representative blots from three experiments with similar results are shown.
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Figure 1: Release of RMCP-1, -5 and CPA from PMC following activation with tc-LIG (PAR-2ap), compound 48/80 and Ag. (A) Supernatants from tc-LIG (10 μM), Ag (10 We/mL) and sham-treated (spon) mast cells were concentrated (10 ×) and Western blot analysis preformed for CPA, RMCP-1 and RMCP-5. Left panel shows Coomassie blue staining of the same gel and right panel Western blot with normal rabbit serum as a negative control. (B) Release of RMCP-1, RMCP-5 and CPA following 20 min and 8 h activation of PMC with tc-LIG (10 μM). (C) Dose response for the release of RMCP-1, -5 and CPA by tc-LIG-stimulated or compound 48/80-stimulated PMC. In all cases representative blots from three experiments with similar results are shown.

Mentions: To identify proteases released by mast cells following PAR-2ap stimulation we activated PMC with tc-LIG (10 μM), and analyzed the supernatants for various mast cell proteases by western blotting, using antisera against the amino-terminal sequences of RMCP-5 and MC-CPA and an antiserum against RMCP-1 protein. In supernatants from tc-LIG-treated PMC one band for RMCP-1 (30 kDa), two bands for RMCP-5 (34 and 35 kDa), and three bands for CPA (40, 41 and 42 kDa) were detected (Fig. 1A). The PAR-2ap tc-LIG induced most of the protease release in the first 20 min. However, proteases accumulated in the conditioned media up to 8 hr (Fig 1B). The release of all three proteases was dose-dependent and was detectable in supernatants of PMC stimulated with tc-LIG at concentrations 0.1 μM and higher (Fig 1C). PMC activation with 48/80 (0.5 mg/ml) induced the release of all three proteases in similar levels to 0.5 μM of tc-LIG (Fig. 1C).


A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation.

Alshurafa HN, Stenton GR, Wallace JL, Hollenberg MD, Befus AD, Vliagoftis H - BMC Pharmacol. (2004)

Release of RMCP-1, -5 and CPA from PMC following activation with tc-LIG (PAR-2ap), compound 48/80 and Ag. (A) Supernatants from tc-LIG (10 μM), Ag (10 We/mL) and sham-treated (spon) mast cells were concentrated (10 ×) and Western blot analysis preformed for CPA, RMCP-1 and RMCP-5. Left panel shows Coomassie blue staining of the same gel and right panel Western blot with normal rabbit serum as a negative control. (B) Release of RMCP-1, RMCP-5 and CPA following 20 min and 8 h activation of PMC with tc-LIG (10 μM). (C) Dose response for the release of RMCP-1, -5 and CPA by tc-LIG-stimulated or compound 48/80-stimulated PMC. In all cases representative blots from three experiments with similar results are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC503387&req=5

Figure 1: Release of RMCP-1, -5 and CPA from PMC following activation with tc-LIG (PAR-2ap), compound 48/80 and Ag. (A) Supernatants from tc-LIG (10 μM), Ag (10 We/mL) and sham-treated (spon) mast cells were concentrated (10 ×) and Western blot analysis preformed for CPA, RMCP-1 and RMCP-5. Left panel shows Coomassie blue staining of the same gel and right panel Western blot with normal rabbit serum as a negative control. (B) Release of RMCP-1, RMCP-5 and CPA following 20 min and 8 h activation of PMC with tc-LIG (10 μM). (C) Dose response for the release of RMCP-1, -5 and CPA by tc-LIG-stimulated or compound 48/80-stimulated PMC. In all cases representative blots from three experiments with similar results are shown.
Mentions: To identify proteases released by mast cells following PAR-2ap stimulation we activated PMC with tc-LIG (10 μM), and analyzed the supernatants for various mast cell proteases by western blotting, using antisera against the amino-terminal sequences of RMCP-5 and MC-CPA and an antiserum against RMCP-1 protein. In supernatants from tc-LIG-treated PMC one band for RMCP-1 (30 kDa), two bands for RMCP-5 (34 and 35 kDa), and three bands for CPA (40, 41 and 42 kDa) were detected (Fig. 1A). The PAR-2ap tc-LIG induced most of the protease release in the first 20 min. However, proteases accumulated in the conditioned media up to 8 hr (Fig 1B). The release of all three proteases was dose-dependent and was detectable in supernatants of PMC stimulated with tc-LIG at concentrations 0.1 μM and higher (Fig 1C). PMC activation with 48/80 (0.5 mg/ml) induced the release of all three proteases in similar levels to 0.5 μM of tc-LIG (Fig. 1C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Alberta, Edmonton, AB, Canada, T6G 2S2. hashem@thrombosis.hhscr.org

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus