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

Our plain-English summary of 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, by A. Fazzini, V. D'Antongiovanni, L. Giusti, et al (PloS One, 2014).

The study

• Authors: A. Fazzini, V. D'Antongiovanni, L. Giusti, Y. Da Valle, F. Ciregia, I. Piano, A. Caputo, A.M. D'Ursi, C. Gargini, A. Lucacchini, M.R. Mazzoni
• Journal: PloS One
• Published: 2014;9(11):e111550
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This was a laboratory study of human cells grown in dishes, not of asbestos exposure or patients. The authors compared a receptor called PAR1, which responds to the clotting enzyme thrombin, in a nonmalignant pleural mesothelial cell line (Met-5A) and a malignant pleural mesothelioma cell line (NCI-H28) that lacks the beta-catenin gene. NCI-H28 cells had more PAR1 than Met-5A and human primary mesothelial cells. Three other mesothelioma cell lines (REN, Ist-Mes2, Mero-14) had levels essentially similar to Met-5A. Thrombin increased growth of both main cell lines, most at 1 nM in Met-5A and at 50 nM in NCI-H28. NCI-H28 and REN cells had less PAR1 on the cell surface. In NCI-H28, two signaling routes were weakened. For example, 10 nM thrombin raised RhoA activation 2.5-fold in Met-5A but only 1.2-fold in NCI-H28. A third route, through Gi proteins, was maintained. Restoring beta-catenin in NCI-H28 cells did not increase surface PAR1, and reducing it in Met-5A caused no significant decrease. The authors concluded that PAR1 in NCI-H28 cells is over-expressed but defective in surface localization and signaling.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-line experiment
• Who was studied: Human cell lines: nonmalignant pleural mesothelial cells (Met-5A) as control; malignant pleural mesothelioma cells NCI-H28, plus REN, Ist-Mes2 and Mero-14; human adult primary mesothelial cells; studied at the University of Pisa, Italy
• Exposure: No asbestos exposure was studied. Cells were treated in the laboratory with thrombin and synthetic PAR1-activating peptides. Beta-catenin was also restored in NCI-H28 cells or reduced in Met-5A cells.
• Main results:
  • PAR1 mRNA was significantly higher in NCI-H28 cells than in Met-5A cells.
  • PAR1 protein was significantly higher in NCI-H28 cells than in primary mesothelial and Met-5A cells. In REN, Ist-Mes2 and Mero-14 cells, levels were essentially similar to Met-5A.
  • Thrombin increased proliferation of both cell lines. The response was maximal at 1 nM in Met-5A cells and at 50 nM in NCI-H28 cells.
  • NCI-H28 cells, and also REN cells, had significantly less cell-surface PAR1 than Met-5A cells.
  • Calcium signaling (the Gq pathway) in response to thrombin or the PAR1 peptide was reduced in NCI-H28 cells compared with Met-5A cells.
  • RhoA activation (the G12/13 pathway) after 10 nM thrombin rose 2.5-fold in Met-5A cells but only 1.2-fold in NCI-H28 cells.
  • The Gi pathway was maintained in NCI-H28 cells. Thrombin inhibited cAMP production by 50% at 1 nM and by approximately 70% (maximal) at 100 nM.
  • ERK1/2 activation by thrombin was maximal at 0.1 nM in Met-5A cells and at 10 nM in NCI-H28 cells.
  • Restoring beta-catenin in NCI-H28 cells did not increase cell-surface PAR1. Reducing beta-catenin with siRNA in Met-5A cells caused no significant decrease in cell-surface PAR1.
• The authors' conclusion: The authors concluded that in NCI-H28 cells PAR1 is over-expressed but defective in cell-surface localization and in signaling through the Gq and G12/13 pathways. They called for further studies of the role of proteases in PAR1 activation and mesothelioma growth.
• Limitations the authors state:
  • The authors did not know whether the increased intracellular PAR1 in NCI-H28 cells was due to altered cell-surface recruitment or to increased internalization of activated receptor.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Homozygous Deletion, Inflammation, Metastasis, Mesothelial Cells, MeT-5A Cells, Mesothelioma.

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