Granzyme K Activates PAR-1 Figure 5. Western blot analysis of effect of GrK on ERK1/2 and p38 MAPK phosphorylation. Cells were incubated with GrK (200 nM) for 10 min, 30 min, or 2 h. Cell lysates were collected and screened for (A) ERK1/2 phosphorylation using anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies and (C) p38 MAPK phosphorylation using anti-phospho-p38MAPK and total p38MAPK antibodies. Cells incubated with ERK1/2 inhibitor (U0126; 10 mM) or p38MAPK inhibitor (SB202190; 10 mM) prior to treatment with GrK (200 nM) for 10 min are included to demonstrate the activity of each inhibitor. Densitometry analysis of immunoblots was carried out using Li-COR Odyssey Infrared imaging system (Li-COR biosciences). (B) Relative levels of ERK1/2 phosphorylation (ERK1 white bars; ERK2 black bars) are expressed as a ratio of phospho-ERK1/2 to total ERK1/2. (D) Relative levels of p38 MAPK phosphorylation are expressed as a ratio of phospho-p38 MAPK to total p38 MAPK. Protein loading was normalized using b-tubulin. The values shown are mean +/2 SEM from three separate experiments. * p,0.05 compared to media alone. doi:10.1371/journal.pone.0021484.g005 GrK-induced proliferation is occurring through a PAR-1 medi- ated mechanism (B). Discussion In this study we demonstrate that GrK can induce pro- inflammatory cytokine release from human lung fibroblasts through the activation of PAR-1 and supports previous studies that have suggested that GrK is not involved in immune-mediated cytotoxity but rather in the promotion of inflammation [3,7,40,41]. Previous work by Joeckel et al [7] demonstrated that GrK induces IL-1b production and secretion from mouse peritoneal macrophages. The IL-1b processing observed in this study was dependent on both the concentration of GrK and its delivery into cells by streptolysin O (SLO). Interestingly, the authors noted that high concentrations of GrK (600–1000 nM) resulted in IL-1b production in the absence of SLO. Whether GrK was exerting this effect via an extracellular or intracellular mechanism was not established. Our study not only demonstrates that GrK can promote inflammation through IL-6, IL-8 and MCP-1 production but also provides evidence that GrK can act extracellularly to exert this function in the absence of a cell delivery agent. Given the tryptase-like activity of GrK, and the role that PARs play in inflammation, it is not unreasonable to hypothesize that in conditions of elevated, free GrK it could cleave and activate PAR-1 and promote inflammation. PAR-1 is involved in a variety of biological events and is the primary receptor responsible for mediating many of the pro- inflammatory and pro-fibrotic effects of thrombin [38,39,42]. PAR-1 activation triggers the production of several inflammatory mediators, including IL-6, IL-8 and MCP-1, and induces cell proliferation through the coupling of receptor activation to one or more intracellular signaling pathways [26,27]. In the present work, neutralizing antibodies and receptor desensitization experiments suggest that GrK induced IL-6, IL-8 and MCP-1 production is dependent on PAR-1 activation through a pathway involving ERK1/2 and p38 MAPK. Furthermore, as is seen with thrombin, GrK-induced IL-6, IL-8 and MCP-1 production is also dependent on ERK1/2 and p38MAPK activation [38,39]. While we focused on ERK1/2 and p38 MAPK signaling in this study, GCPRs can also initiate other signaling cascades pathways (reviewed in [26,27,29]). Recently, Deng et al (2008) demonstrated that MCP-1 release in murine lung fibroblasts was regulated by multiple pathways including phospholipase C, calcium-dependent PKC, and Rho kinase signaling pathways [37]. GrK-induced PAR-1 activation is likely associated with multiple signaling pathways and further biochemical analysis is required to delineate which signaling pathways are involved. PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21484