Figures & data
FIGURE 1 Cells were either treated with 2 U/mL thrombin for 4 hours with or without pretreatment with 60 mM PAR-1 siRNA or 1 μM argatroban for 30 minutes. A549 cells were transfected with PAR-1 siRNA using transfection reagent for 6 hours at 37°C, washed using 2x normal growth media containing antibiotics and incubated in 1x normal growth media. Cells were also treated with 300 μM TFLLR for 4 hours for real-time PCR (A). PAR-1 protein levels were determined by immunoblotting after same treatments for 72 hours (B). Thrombin and PAR-1 activating peptide, TFLLR increased PAR-1 mRNA expression in A549 cells by quantitative real-time PCR. PAR-1 siRNA transfection or pre-treatment of thrombin inhibitor, argatroban, suppressed thrombin-induced PAR-1 mRNA expression (A). Thrombin and TFLLR increased PAR-1 protein expression as assessed by Western blot. PAR-1 siRNA transfection or pretreatment with argatroban inhibited thrombin-induced PAR-1 protein expression (B). Data are presented as means ± SE; n = 5/group. *, † P < .05; **P < .01. *, **; compared with control. †; compared with thrombin.
FIGURE 2 Cells were either treated with 2 U/ml thrombin for 4 hours with or without pretreatment with 60 mM PAR-1 siRNA or 1 μM argatroban for 30 minutes. A549 cells were transfected with PAR-1 siRNA using transfection reagent for 6 hours at 37°C, washed using 2x normal growth media containing antibiotics and incubated in 1x normal growth media. Cells were also treated either with 300 μM TFLLR or 10 ng/mL TGF-β for 4 hours for real-time PCR. Thrombin and TFLLR increased α-SMA and collagen I mRNA expression and suppressing E-cadherin mRNA expression by quantitative real-time PCR. PAR-1 siRNA or pretreatment with argatroban inhibited thrombin-induced α-SMA and collagen I mRNA expression, and reversed thrombin-induced suppression of E-cadherin mRNA expression. Data are means ± SE; n = 5/group. *, † P < .05; ** P < .01. *, **; compared with control. †, ††; compared with thrombin.
FIGURE 3 Cells were either treated with 2 U/mL thrombin for 72 hours with or without pretreatment with 60 mM PAR-1 siRNA or 1 μM argatroban for 30 minutes. A549 cells were transfected with PAR-1 siRNA using transfection reagent for 6 hours at 37°C, washed using 2x normal growth media containing antibiotics and incubated in 1x normal growth media. Cells were also treated either with 300 μM TFLLR or 10 ng/mL TGF-β for 72 hours for immunoblotting. Thrombin and TFLLR increased α-SMA and collagen I protein expression while PAR-1 siRNA transfection or pretreatment with argatroban reversed thrombin-induced α-SMA and collagen I expression as assessed by Western blot. PAR-1 siRNA or pretreatment with argatroban reversed thrombin-induced suppression of E-cadherin expression. Data are means ± SE; n = 5/group. *, † P < .05; **, †† P < .01. *, **; compared with control. †, ††; compared with thrombin.
FIGURE 4 After thrombin treatment (2 U/mL) for 2 hours, A549 cells were harvested by centrifugation (1000 rpm for 5 minutes). Cytosolic and membrane fractions were isolated from the lysed cells using a Mitochondrial/Cytosol Fractionation Kit. Cell lysates were prepared, and then immunoblotted with antibodies for PKCα, PKCδ, and PKCε. Experiments were performed three times, and representative immunoblots are presented. Thrombin increased the migration of PKCα, PKCδ, and PKCε from cytosol to membrane fractions. PAR-1 siRNA or pretreatment with argatroban inhibited thrombin-induced PKCα, PKCδ, and PKCε activation. Addition of GÖ6976 (10 nM) to thrombin inhibited migration of PKCα; addition of rottlerin (4 μM)-inhibited migration of PKCδ; PKCε antagonist peptide (10 μM) inhibited migration of PKCε.
FIGURE 5 Expression of E-cadherin, α-SMA, and collagen I in cultured A549 cells was assessed with Western blot. Cells were either treated with 2 U/mL thrombin for 72 hours with or without pretreatment with 60 mM PAR-1 siRNA or 1 μM argatroban or 10 nM GÖ6976 or 4 μM rottlerin or 10 μM PKCε antagonist peptide for 30 minutes. Cells were also treated with 10 ng/mL TGF-β for 72 hours for positive control. Cell lysates were prepared, and then immunoblotted with antibodies for E-cadherin, α-SMA, and collagen I. Thrombin significantly increased α-SMA and collagen I expression and decreased E-cadherin expression. PAR-1 siRNA transfection, pretreatment with argatroban, GÖ6976, rottlerin or PKCε antagonist peptide inhibited epithelial-to-mesenchymal transition by thrombin. Data are means ± SE; n = 5/group. *, † P < .05; **, †† P < .01. **; compared with control. †, ††; compared with thrombin.
FIGURE 6 A549 alveolar epithelial-to-myofibroblast transition in vitro. Immunoreactivity for both α-SMA and E-cadherin was assessed by immunofluorescence after stimulation with thrombin, TFLLR, and TGF-β. (A) Dual immunocytochemical staining showed that thrombin, TFLLR, and TGF-β decreased cadherin (green) and increased α-SMA (red) expression in cultured A549 cells. (B) Dual immunocytochemical staining showed that PAR-1 siRNA or pretreatment with argatroban, GÖ6976, rottlerin, or PKCε antagonist peptide inhibited epithelial-to-mesenchymal transition induced by thrombin. All recovered cadherin (green) expression and inhibited α-SMA expression (red) induced by thrombin in cultured A549 cells. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (blue). Original magnification, ×1000. All experiments were performed three times, and representative images are presented. Merge indicates sum of DAPI, E-cadherin, and α-SMA. (C) Typical microphotographs of A549 cells stimulated with the thrombin. Addition of thrombin especially at a 2.0 U/mL concentration for 72 hours changed the polygonal A549 cells to a more elongated mesenchymal phenotypes. Original magnification, ×400. (Continued)
FIGURE 7 (A) Phosphorylation of extracellular signal-regulated kinase ERK1/2 by thrombin in A549 cells. After thrombin treatment for 2 hours, whole cell extracts were prepared and 50 μg of protein subjected to SDS-PAGE and Western blot. Blots were probed with antibodies against phosphorylated ERK (p-ERK1/2) and total ERK2. PAR-1 siRNA or pretreatment with argatroban, PKCα/β inhibitor (GÖ6976), PKCδ inhibitor (rottlerin), or PKCε antagonist peptide inhibited ERK1/2 phosphorylation activated by thrombin. *, † P < .05, **, †† P < .01 (n = 6). *, **; compared with control. †; compared with thrombin. (B) Thrombin induces type I collagen and TGF-β release. A549 cells were incubated with thrombin for 72 hours or with other pretreatments; PAR-1 siRNA, argatroban, and PKC inhibitors. Collagen I and TGF-β concentrations were measured by ELISA in supernatants of cell culture. Type I collagen and TGF-β were significantly increased by thrombin compared with the blank control. Effects were attenuated by PAR-1 siRNA, pretreatment with thrombin inhibitor argatroban, PKCα/β inhibitor GÖ6976, PKCδ inhibitor rottlerin, or PKCε antagonist peptide. *, † P < .05, **, †† P < .01. *, **; compared with control. †, ††; compared with thrombin (n = 4).
