Granzyme K Activates PAR-1 Figure 4. Desensitization of PAR-1 with Thrombin (2.5 U/ml) reduces GrK-induced IL-6, IL-8 and MCP-1 production in lung fibroblasts. (A–C) HFL were treated with thrombin (2.5 U/ml) for 10 min, monolayers were then rinsed three times with fresh media and incubated with GrK (200 nM) for 24 h. Supernatants were collected and IL-6, IL-8 and MCP-1 levels were analyzed by ELISA. Supernatants were normalized using cell counts. Data are expressed as % change over media control 6 SEM from three separate experiments run in triplicate. (D) GrK (200 nM) and thrombin (2.5 U/ml) induced ERK1/2 phosphorylation following 10 min incubation. Pre-treatment (PT) of HFL with thrombin (2.5 U/ml) for 10 min prior to treatment with GrK (200 nM) for an additional 10 min decreased ERK1/2 phosphorylation when compared to monolayers incubated with GrK (200 nM) alone. ERK1/2 phosphorylation was assessed by Western blotting of cell lysates using anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies. Protein loading was verified and normalized using b-tubulin. Data represent the mean 6 SEM of at least three independent experiments. + p,0.05 when compared to media control (1% DMSO in DMEM); ** p,0.05 compared to GrK (200 nM) treated cells. doi:10.1371/journal.pone.0021484.g004 shown in Figure 4 (A–C), an initial proteolytic activation of PAR-1 by thrombin prior to incubation with GrK decreased IL-6, IL-8 and MCP-1 production. This was further confirmed by examina- tion of ERK1/2 phosphorylation where pre-treatment (PT) with thrombin decreased the level of phosphorylation elicited by subsequent application of GrK (Figure 4 D). GrK induces ERK1/2 and p38 MAPK phosphorylation in HFL It has previously been reported that activation of PAR-1 leads to the activation of ERK1/2 and p38 MAPK in several cell types, including fibroblasts [37,38,39]. Thus, we next performed experiments to determine if GrK activation of PAR-1 induced ERK1/2 or p38MAPK phosphorylation in HFL. Figure 4 A–D demonstrates that incubation with GrK (200 nM) induced phosphorylation of both ERK1/2 and p38MAPK. Phosphoryla- tion of ERK1/2 was maximal 10 min after stimulation with GrK and gradually declined to control levels by 2 h incubation (Figure 5 A and B). GrK also induced p38 MAPK phosphorylation with a similar kinetic profile (Figure 5 C and D). MAPK activation in PAR-mediated IL-6, IL-8 and MCP-1 release Using specific pharmacological inhibitors, we assessed the relative roles of ERK1/2 and p38 MAPK activation on the synthesis and release of GrK- and thrombin-induced IL-6, IL-8 and MCP-1 in HFL. As shown in Figure 5, pre-treatment of cells with the MEK1/2 inhibitor U0126 inhibited GrK-induced IL-6 and IL-8 release, and thrombin-induced IL-6, IL-8 and MCP-1 release back to control levels. While a reduction was noted in GrK-induced MCP-1 levels, levels were not significantly different from controls (Figure 6 C). In contrast, inhibition of p38 MAPK with SB202910 (10 mM) had no significant impact on GrK- induced IL-6, IL-8 or MCP-1 production but did significantly reduce thrombin-induced IL-6, IL-8 and MCP-1 levels, suggesting that although GrK can activate p38 MAPKs in HFL, p38 may not be involved in mediating PAR-1 induced IL-6, IL-8 or MCP-1 production. GrK stimulates HFL proliferation Because fibroblasts are a major component of connective tissues and fibroblasts proliferation is an important process in wound healing, we examined the functional consequences of GrK- induced signaling on fibroblast viability and proliferation. As can be seen in Figure 7, GrK stimulated proliferation in a concentration dependent manner (A). No significant difference was detected between controls and GrK-treated cells prior to 48 h (data not shown). GrK (200 nM)-induced proliferation was inhibited by ATAP-2 (5 mg/ml) (black bars) suggesting that PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21484