Figure 1.
NCI-H28 cells over-express PAR1.
A, relative expression levels of PAR1 mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR1 protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR1 expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.
Figure 2.
PAR1 agonists enhance Met-5A and NCI-H28 cell proliferation.
Met-5A and NCI-H28 cells were serum and growth factor starved for 18 h and then treated with different concentrations of agonists for 72 h. Cell proliferation was measured using a mitochondrial activity assay (WST-1). The optical density values of vehicle treated Met-5A and NCI-H28 cells (Ctrls) were 0.210±0.03 and 0.232±0.04 (n = 6; ns by Student’s t test), respectively. A, thrombin-induced cell proliferation (n = 6); B, non-selective PAR1-AP-induced cell proliferation (n = 6); C, selective PAR1-AP-induced cell proliferation (n = 3). Data shown are mean ± SEM of at least three independent experiments performed in triplicate. The differences in proliferation between Ctrl and agonist-treated cells were significant (*P≤0.05, **P≤0.01, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Figure 3.
NCI-H28 and REN cells express significant less amount of PAR1 on plasma membrane.
Cell surface PAR1 expression was measured by ELISA using a polyclonal antibody which recognizes the N-terminus of PAR1 [31]. Antibody binding to fixed cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR1 expression between Ctrl (Met-5A cells) and MPM (NCI-H28 and REN) cellswere significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Figure 4.
PAR1 agonist-induced Gq but not Gi signaling is impaired in NCI-H28 cells.
A, thrombin-induced intracellular Ca2+ mobilization in HMEC-1, Met-5A, and NCI-H28 cells. B, selective-PAR1-AP-induced intracellular Ca2+ mobilization in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were loaded with Fluo-3AM to measure [Ca2+]i variations as indicated by changes in fluorescence intensity. Fluorescence was recorded before agonist addition (F0) and then every 3 seconds after thrombin (10 nM) or PAR1-AP (10 µM) addition for another 120 seconds. Data shown are mean ± SEM of a single experiment done in triplicate. Experiments were repeated two additional times with similar results. The results are reported as relative fluorescence (RF = F/F0 where F0 is basal fluorescence and F is fluorescence recorded after cell stimulation with the agonist). C, inhibition of isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells by different concentrations of thrombin in the presence and absence of 100 nM SCH 79797. D, no effect of the selective PAR1-AP on isoproterenol stimulated cAMP production in Met-5A and NCI-H28 cells. Serum and growth factor starved cells were exposed to different agonist concentrations. Assays were initiated by the addition of 1 µM isoproterenol. Production of cAMP was measured using a competition binding assay which includes the bovine adrenal cAMP binding protein and [3H]cAMP. Data shown are mean ± SEM of three independent experiments performed in triplicate. The differences between thrombin- and thrombin plus SCH 79797-treated cells were significant (**P≤0.01, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).
Figure 5.
PAR1 agonist-induced G12/13 signaling is impaired in NCI-H28 cells.
A, relative levels of RhoA activation in response to thrombin in Met-5A and NCI-H28 cells. B, relative levels of RhoA activation in response to the selective PAR1-AP in Met-5A and NCI-H28 cells. Rho A activation was measured in serum and growth factor starved cells using the RhoA G-LISA kit from Cytoskeleton. Data shown are mean ± SEM of three independent experiments performed in triplicate. The differences in RhoA activation between Ctrl (vehicle treated Met-5A or NCI-H28 cells) and agonist-treated cells were significant (*P≤0.05, **P≤0.01, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).
Figure 6.
Thrombin differently induces ERK1/2 activation in Met-5A and NCI-H28 cells.
A, relative intensity of pERK1/2 immunoreactive bands quantified by densitometric scanning. Serum and growth factor starved Met-5A and NCI-H28 cells were incubated in the presence and absence of various thrombin concentrations ranging from 0.01 to 100 nM for 5 min. ERK1/2 activation was then determined using a specific anti-phospho-ERK1/2 antibody. Nitrocellulose membranes were then stripped and reprobed for total ERK1/2. Data (mean ± SEM) are expressed as fold-increase over Ctrl and are the averages of three independent experiments performed in duplicate. The differences in phosphorylated ERK1/2 level between Ctrl (vehicle treated Met-5A or NCI-H28 cells) and thrombin-treated cells were significant (*P≤0.05, **P≤0.01) by one-way ANOVA followed by Bonferroni’s multiple comparison test. B, a representative immunoblot.
Figure 7.
Cellular distribution of caveolin-1 and PAR1 in Met-5A and NCI-H28 cells.
Immunolabeling of β-catenin, caveolin-1 and PAR1 in Met-5A and NCI-H28 cells was performed as described in Materials and Methods. The images shown are representative of many cells examined in two independent experiments. The arrows point out intracellular or plasma membrane localization of immunostained proteins. Scale Bar: 10 µm.
Figure 8.
Double immunofluorescence labelling of caveolin-1 and PAR1 in Met-5A and NCI-H28 cells.
Double labelling was performed by incubating antibodies as follow: anti-PAR1 and anti-caveolin-1; anti-PAR1 and rabbit polyclonal anti-β-catenin; mouse monoclonal anti-β-catenin and anti-caveolin-1. To visualize double fluorescent staining, cells were incubated with Alexa Fluor 488- and Alexa Fluor 568-labeled goat anti-mouse and anti-rabbit antibodies as described in Materials and Methods. The images shown are representative of many cells examined in two independent experiments. The yellow stain indicates protein proximity (see arrows). All images were analyzed using the ImageJ program [34]. PCC values are expressed as mean ± SEM of six examined area. PCC value for PAR1/caveolin-1 colocalization was 0.77±0.05 and 0.84±0.03 in Met-5A and NCI-H28 cells, respectively. PCC values for PAR1/β-catenin and caveolin-1/β-catenin colocalization in Met-5A cells were 0.70±0.02 and 0.55±0.04, respectively. Scale Bar: 10 µm.
Figure 9.
Neither β-catenin rescue nor deletion modify cell surface PAR1 expression.
NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR1 expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR1 expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).