receptor for trypsin and mast cell tryptase, and PAR-3 and PAR-4 are receptors for thrombin [27,28,29]. In the present study, we investigated whether extracellular GrK could induce PAR activation in human lung fibroblasts. GrK induced the production of interleukin-6 (IL-6), IL-8 (CXCL8) and monocyte chemotactic protein-1 (MCP-1)/chemokine c-c motif ligand 2 (CCL2) in human lung fibroblasts through the activation of PAR-1. In addition, GrK induced fibroblast proliferation in a PAR-1-dependent manner suggesting that elevated extracellular GrK could augment inflammation and play a role in airway remodeling through the activation of PAR-1. Materials and Methods Reagents Cell culture medium Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and PBS were obtained from Invitrogen (Carlsbad, CA, USA). Thrombin, ERK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB202190, and the antibiotics (penicillin and streptomycin) were obtained from Sigma (St. Loius, MO, USA). Granzyme K was obtained from Axxora (Burlington, ON Canada), and the PAR-1 (ATAP-2 cat no: sc- 13503) and PAR-2 (Sam11L cat no: sc-13504) neutralizing antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Mouse IgG (Abcam cat no 37355) was used as an isotype control. Antibodies: Phospho-p44/42 (Cell Signaling Technology (clone E10) cat no: 9106s), total p44/42 (Cell Signaling Technology (clone 137F5) cat no: 4695), phospho-p38 MAPK (Cell Signaling Technology (clone 28B10) cat no: 9216), total p38 MAPK (Cell Signaling Technology cat no: 9212s) b- tubulin (Millipore (clone AA2) cat no: 05-661) was purchased from Millipore, goat anti-mouse IRD 700 and goat IRD anti-rabbit 800 were purchased from LI-COR Biotechnology (Lincoln, Nebraska). IL-6, IL-8 and MCP-1 Duoset ELISAs were purchased from R&D Technologies (Burlington, ON, Canada). Dimethyl sulphoxide (DMSO) was purchased from Sigma (St. Loius, MO, USA) and sodium azide was purchased from sigma (St. Louis, MO, USA). Cell lysis solution, protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Sigma (St. Loius, MO, USA). BioRad protein reagent and nitrocellulose membranes were purchased from Bio-Rad (Mississauga, ON, Canada). Cell Culture Human fetal lung fibroblasts (HFL) were purchased from ATTC (Cat. No CCL-153). Cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 mg/ml penicillin, 100 mg/ml streptomycin, and kept in a humidified atmosphere with 5% CO2. All experiments were performed between passages 5–11. Cells were routinely passaged every 4–5 d. For all experiments, cells were grown to confluency and serum-starved in DMEM with 0.1% FBS for 24 h before treatment. Detection of IL-6, IL-8, MCP-1 IL-6, IL-8 and MCP-1 levels were determined using human IL- 6, IL-8 or MCP-1 ELISAs, according to according to manufac- turer’s protocols. Briefly, cells were seeded in 12-well culture dishes (16106 per well), grown to 90% confluency and serum-starved in DMEM (0.01% FBS) over-night. Cells were then incubated with GrK (10–300 nM), in the absence of any delivery/cell permea- bilizing agent (ie. perforin, streptolysin O), or Thrombin (2.5 U/ ml), at the concentrations and time points indicated for each experiment in DMEM containing 1% FBS. Supernatants were collected for analysis by ELISA and the remaining cells were trypsinized and counted using a hemocytometer. For inhibitor Granzyme K Activates PAR-1 studies, cells were pre-treated with the ERK inhibitor (U0126; 10 mM) or the p38 MAPK inhibitor (SB202190; 10 mM) or neutralizing antibodies for 45 min prior to stimulation with GrK (200 nM) or thrombin (2.5 U). Cells treated with vehicle alone, heat-inactivated GrK (100 nM, heat-inactivated at 85uC for 5 min) or mouse IgG (5 mg/ml) isotype served as baseline controls. Western blot analysis of signal transduction pathways in HFLs upon GrK stimulation HFL were seeded in 6-well culture dishes at 26106 cells/well, grown to desired confluency, serum-starved over night and then treated with 10 mM U0126 or 10 mM SB202910 for 45 min prior to treatment with 200 nM of GrK or 2.5 U/ml of Thrombin. After 10 min, 30 min, and 2 h, supernatants were collected for IL- 6, IL-8 and MCP-1 determination and remaining cells harvested for Western blot analysis of the levels of the downstream products for phospho-p44/42 (ERK1/2) and phospho-p38 MAPK. Briefly, cells were rinsed with ice-cold Dulbecco’s phosphate- buffered saline (dPBS) and lysed in 100 ml of cell lysis buffer containing proteinase inhibitor cocktail and phosphatase-inhibitor cocktail followed by scraping with a cell scraper. Cell debris was removed by centrifugation (12,0006 g for 15 min) and protein was quantified by the Bradford method (BioRad) using bovine serum albumin as standard. Samples containing equal amounts of total cell protein were separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane using the Bio-Rad wet transfer system at 100 v for 1 h. The membranes were blocked with 2.5% skim milk in TBST (50 mM Tris, pH 7.6, 0.15 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Membranes were then incubated with antibodies against phospho-p44/42 MAPK (1:2000), total p44/42 MAPK (1:1000), phospho-p38 MAPK (1:2000), p38 MAPK (1:1000) over night at 4uC with gentle shaking. After several washes in TBST, membranes were incubated with goat anti-mouse IRD 700 and goat IRD anti- rabbit 800 for 1 h at room temperature. Flourescent signal was imaged using the Li-COR Odyssey Infrared imaging system (Li- COR biosciences). Densitometry was used to quantify all bands. Membranes were then re-probed with mAb b-tubulin (1:5000) for 1 h at room temperature and incubated with goat anti-mouse IRD 800 for 45 min. The relative levels of phospho-p44/42 MAPK or phospho-p38MAPK are expressed as the ratio to b- tubulin. PAR-1 desensitization For PAR-1 receptor desensitization studies, cells were pre-treated with 2.5 U/ml of thrombin for 10 min. Media was removed and cells were washed three times with PBS. For IL-6, IL-8 and MCP-1 measurements, fresh media containing GrK (200 nM) or thrombin (2.5 U/ml) was then added cells for a 24 h incubation period and supernatants were collected and screened as described above. In addition to measuring potential changes in cytokine and chemokine production, thrombin-induced PAR-1 activation will induce ERK1/2 phosphorylation. Therefore, we screened for changes in p44/42 (ERK1/2) activation following receptor de-sensitization in cells were pre-treated with thrombin. Here cells were pre-treated with thrombin (2.5 U/ml) for 10 min, media was removed and cells were washed three times with PBS. Cells were then treated with either GrK (200 nM) or thrombin (2.5 U/ml) for 10 min. Cell lysates were then collected as described above and screened for phospho-p44/42 activation as described. Cell viability and proliferation As extracellular GrA and GrB have been shown to induce cell detachment, we examined the impact of extracellular GrK on cell PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21484