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Altered protease-activated receptor-1 expression and signaling in a malignant pleural mesothelioma cell line, NCI-H28, with homozygous deletion of the β-catenin gene.

Fazzini A, D'Antongiovanni V, Giusti L, Da Valle Y, Ciregia F, Piano I, Caputo A, D'Ursi AM, Gargini C, Lucacchini A, Mazzoni MR - PLoS ONE (2014)

Bottom Line: We found that the expression level of PAR1 was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells.We also determined that PAR1 signaling through Gq and G12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells.Thus, our results provide evidences for dysfunctional PAR1 signaling in NCI-H28 cells together with reduced plasma membrane localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.

ABSTRACT
Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR1 in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR1 was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR1 over-expression compared to Met-5A cell line. Thrombin and PAR1 activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR1 signaling through Gq and G12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR1 signaling through Gi proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR1 expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR1 signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR1 in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.

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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).
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pone-0111550-g004: 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).

Mentions: To further explore PAR1 ability of signaling in the NCI-H28 cell line, receptor-activated Gq, Gi, and G12/13 signaling pathways were examined. First, we investigated PAR1-activated Gq signaling by analyzing intracellular Ca2+ mobilization after cell stimulation with either thrombin or the selective PAR1-AP. As indicated by relative fluorescence increase, both thrombin (10 nM) and PAR1-AP (10 µM) induced rapid and transient increase of [Ca2+]i in Met-5A as well as in HMEC-1 as previously reported (Figure 4.A and 4.B) [32]. In contrast, thrombin- or PAR1-AP-stimulation of NCI-H28 cells resulted in a reduced increase of [Ca2+]i (Figure 4.A and 4.B). Given the sharply contrasting results, we examined both cell lines for the expression levels of some components of the Gq signaling pathway by immunoblot analysis (Figure S1). Whereas PLC-β1 was expressed at similar levels in both cell lines, the amount of Gαq was apparently greater in NCI-H28 than Met-5A cells (Figure S1).


Altered protease-activated receptor-1 expression and signaling in a malignant pleural mesothelioma cell line, NCI-H28, with homozygous deletion of the β-catenin gene.

Fazzini A, D'Antongiovanni V, Giusti L, Da Valle Y, Ciregia F, Piano I, Caputo A, D'Ursi AM, Gargini C, Lucacchini A, Mazzoni MR - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4218765&req=5

pone-0111550-g004: 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).
Mentions: To further explore PAR1 ability of signaling in the NCI-H28 cell line, receptor-activated Gq, Gi, and G12/13 signaling pathways were examined. First, we investigated PAR1-activated Gq signaling by analyzing intracellular Ca2+ mobilization after cell stimulation with either thrombin or the selective PAR1-AP. As indicated by relative fluorescence increase, both thrombin (10 nM) and PAR1-AP (10 µM) induced rapid and transient increase of [Ca2+]i in Met-5A as well as in HMEC-1 as previously reported (Figure 4.A and 4.B) [32]. In contrast, thrombin- or PAR1-AP-stimulation of NCI-H28 cells resulted in a reduced increase of [Ca2+]i (Figure 4.A and 4.B). Given the sharply contrasting results, we examined both cell lines for the expression levels of some components of the Gq signaling pathway by immunoblot analysis (Figure S1). Whereas PLC-β1 was expressed at similar levels in both cell lines, the amount of Gαq was apparently greater in NCI-H28 than Met-5A cells (Figure S1).

Bottom Line: We found that the expression level of PAR1 was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells.We also determined that PAR1 signaling through Gq and G12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells.Thus, our results provide evidences for dysfunctional PAR1 signaling in NCI-H28 cells together with reduced plasma membrane localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.

ABSTRACT
Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR1 in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR1 was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR1 over-expression compared to Met-5A cell line. Thrombin and PAR1 activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR1 signaling through Gq and G12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR1 signaling through Gi proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR1 expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR1 signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR1 in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.

Show MeSH
Related in: MedlinePlus