Coughlin SR

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Coughlin SR. Several shortened versions of the PAR1-derived and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. Conclusions: The results indicate that both APC and particular PAR1-derived and PAR3-derived peptides which are biased agonists for PAR1 or PAR3 can reduce inflammasome activity in stimulated human being monocytes as measured by caspase-1 activity and IL-1 launch and that PAR-derived biased peptide agonist mixtures are synergistically anti-inflammatory. and reduces vascular leakage [18,21C25]. P1(47C66) at 50 M as well as at several lower concentrations reduced caspase-1 activity, whereas neither a scrambled P1(47C66) peptide nor the peptides, SFLLRN or TFLLRN, which resemble the novel N-terminal amino acid sequence generated by thrombins PAR1 cleavage at Arg41 experienced any effect on caspase Cdh15 activity (Numbers 1A & 1C). Protecting signaling by APC can follow its cleavage of PAR3 at Arg41 on endothelial cells, and the PAR3-derived biased agonist peptide, P3(42C65) or P3R, results in protective effects and reduces neutrophil extracellular capture formation [17,26]. Much like APC and P1(47C66), the P3(42C65) biased agonist peptide reduced caspase activity (Numbers 1B & 1D). Peptide P3(39C65), the PAR3 peptide representing thrombin cleavage at Lys38, in addition to a scrambled P3(42C65) control peptide, did not significantly switch caspase activity in THP-1 cells (Number 1B). Open in a separate window Number 1. APC, a PAR1-derived peptide, and a PAR3-derived peptide each can reduce caspase activity.THP-1-null (THP-1) cells were plated at a final concentration of 1 1 106/mL in 96 well plates and incubated with PMA (0.5 M) for 3 hours at 37C in supplemented RPMI. Press was consequently changed every 24 hours for 3 days. Subsets of wells were selected and cells were treated for 60 moments at 37C in serum-free RPMI with: (A) APC (4 g/mL), P1(47C66) (50 M), scrP1(47C66) (50 M), SFLLRN (50 M), or TFLLRN (50 M); (B) P3(42C65) (50 M), scrP3(42C65) (50 M), or P3(39C65) (50 M); (C) P1(47C66) (2 nM – 50 M); or (D) P3(42C65) (2 nM – 50 M). Following DPBS wash, BIBF0775 cells were incubated with LPS (1 g/mL) for 3 hours at 37C in serum-free RPMI. After a DPBS wash, cells were incubated with ATP (5 mM) in the presence or absence of 10 M YVAD (caspase-1 inhibitor) or 2.5 M ZVAD (pan-caspase inhibitor) for 45 minutes at 37C, prior to carrying out the caspase-1 activity assay following manufacturers directions measuring luminescence (relative luminescent units, RLU). (C) and (D) Experimental caspase-1 activity was normalized to YVAD (caspase-1 inhibitor) luminescence devices. Data points symbolize imply S.D. BIBF0775 of at least 3 self-employed experiments. *P 0.005 (vs. LPS + ATP); #P 0.05 (vs. LPS +ATP); n.s. not significant. While PAR3 itself is generally thought to be a non-signaling receptor, it is speculated that PAR3 BIBF0775 can modulate PAR1 signaling through PAR1:PAR3 dimerization [24,27C29]. Therefore, we hypothesized that although low concentrations of either P1(47C66) or P3(42C65) are insufficient to exert anti-inflammatory activity by reducing caspase activity, the combination of these two GPCR biased agonists could synergistically reduce caspase activity. When P1(47C66) at 10 nM in the presence of 500 nM P3(42C65) was incubated with THP-1 cells prior to induction of caspase activity, a subsequent reduction in caspase activity was observed, in contrast to the effects of P1(47C66) in the absence of P3(42C65) (Number 2A). Furthermore, when P3(42C65) at concentrations 4 nM was incubated with 1 nM P1(47C66), a significant reduction in normalized caspase-1 activity was observed, in contrast to P3(42C65) in the absence of P1(47C66) (Number 2B). In an alternate manner, active caspase-1 was measured using the FAM-FLICA assay, measuring the fluorescently labelled caspase-1 inhibitor YVAD-FMK [2,30]. APC, P1(47C66), P3(42C65) or the peptide combination resulted in a significant decrease in active caspase-1 detected suggesting the reduction in activity of caspase-1 is definitely reflective of a reduction in active enzyme (Number 3A). Next, we sought to determine whether this reduction in caspase-1.