Cox–2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells through generation of prostaglandin-gycerol esters

Abstract

Aims: We have shown previously that the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is a lipid mediator that blocks proliferation and induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death still need to be clarified. AIM: To analyze a possible role of COX–2 in the differential cell death of primary HSCs and hepatocytes induced by 2-AG metabilization to pro-apoptotic prostaglandin D2 glycerol ester (PGD2-GE). Methods: Primary rat hepatocytes and rat HSCs were isolated from healthy rat liver by collagenase perfusion. 2-AG- or PGD2-GE-induced cell death was analyzed by LDH assay and western blot for caspase 3- and PARP-cleavage. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. COX–2 expression was determined by western blotting. Results: HSCs displayed significant COX–2 protein expression, whereas hepatocytes did not express notable levels of COX–2. Treatment of primary culture-activated rat HSCs with 2-AG dose-dependently induced cell death (70% after 24h at 10µM) and was accompanied by PARP- and caspase 3-cleavage. Similarly, PGD2-GE also induced apoptotic cell death dose-dependently in primary HSCs. In contrast, 2-AG did not induce cell death in primary rat hepatocytes at concentrations of up to 100uM, but PGD2-GE lead to marked cell death starting at 25µM. Both, 2-AG- and PGD2-GE induced ROS formation in HSCs, indicating common ROS-dependent pathways to induce cell death. Conclusion: HSCs and hepatocytes differentially express COX–2, leading to differential metabolization of the endocannabinoid 2-AG. The generation of PGD2-GE by COX–2 in HSCs possibly contributes to the different susceptibility of hepatocytes and HSCs towards 2-AG-induced cell death.