Pertussis Toxin, an Inhibitor of Gαi PCR, Inhibits Bile Acid- and Cytokine-Induced Apoptosis in Primary Rat Hepatocytes

Golnar Karimian,Han Moshage,Manon Buist-Homan,Klaas Nico Faber

doi:10.1371/journal.pone.0043156

Golnar Karimian, Han Moshage + Show 2 more

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https://doi.org/10.1371/journal.pone.0043156

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Abstract

Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. GαiPCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting GαiPCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50–200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (−60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of GαiPCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.

Highlights

In chronic and acute liver diseases, the liver is exposed to increased levels of cytokines, reactive oxygen species and bile acids, all of which independently can lead to loss of functional liver mass due to hepatocyte cell death
To investigate whether pertussis toxin (PT) has the same anti-apoptotic effect in a human hepatocellular carcinoma cell line and a rat hepatoma cell line as it has in primary rat hepatocytes, we investigated the effect of PT in HepG2-rNtcp cells and rat H-4-II-E cells respectively
We report that PT, an inhibitor of Ga-proteins, protects primary rat hepatocytes against bile acid- and cytokineinduced apoptosis

Summary

Introduction

In chronic and acute liver diseases, the liver is exposed to increased levels of cytokines, reactive oxygen species and bile acids, all of which independently can lead to loss of functional liver mass due to hepatocyte cell death. Hepatic stellate cells become activated, start proliferating and produce excessive amounts of extracellular matrix proteins leading to liver fibrosis, which may progress to end-stage liver disease [1]. Apoptotic bodies are cleared by surrounding phagocytizing cells that minimize inflammation. Despite worldwide efforts to establish therapeutic strategies for liver injury, end-stage liver disease remains a high burden for public health due to the lack of effective treatments. Excessive hepatocyte apoptosis is often observed in liver disease and, as this is a highly controlled cellular mechanism, drugs and therapeutic strategies to prevent hepatocyte apoptosis may help to maintain sufficient liver mass and function [1]

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