Externalization of phosphatidylethanolamine in hepatocytes from male mice is increased by iPLA2β deficiency associated with cellular oxidative stress

Background and Aims:

Calcium-independent group VIA phospholipase A2 (iPLA2ß) plays an important role in phospholipid remodeling, homeostasis as well as apoptosis. Studies have shown that on endothelial cells and myeloma cells, phosphatidylethanolamine (PE) is externalized to the outer leaflet of the plasma membrane during oxidative stress (Stafford JH, Thorpe PE. Neoplasia. 2011. 13:299 – 308). However, the correlation of iPLA2ß deficiency on PE externalization and its response to oxidative stress in hepatocytes is completely unknown. Previously, we have shown that PE contents in livers of male iPLA2ß-null (KO) mice were increased markedly as compared to wild-type mice, and this was not found in female mutant mice. Hence, we sought to investigate whether PE contents and PE externalization would be increased in iPLA2ß-deficient hepatocytes isolated from male and female mice. We further determined whether PE externalization could be affected by oxidative stress or apoptosis. Methods:

Hepatocytes were isolated from male and female aged-matched control C57BL/6 (WT) and KO mice at 5 – 12 weeks old, and subjected to different treatment for oxidative stress or apoptosis induction. Flow cytometry was carried out to detect externalized PE on hepatocytes by using Duramycin-NHS-Biotin (DLB) as a probe which has a high affinity to PE. DLB was then coupled with AlexaFlour-488 streptavidin for detection. Oxidative stress levels were determined by flow cytometry of dichlorofluorescin (DCFH). Apoptosis injury was measured by luminescence assays of cleaved caspase 3/7 activities.

Results:

iPLA2ß deficiency increased the levels of DLB-streptavidin (+) and DCFH (+) hepatocytes isolated from male but not from female mice. Thus, iPLA2ß-deficient hepatocytes from male mice showed increased externalized PE and oxidative stress. Upon treatment with 50µM menadione (redox cycling to generate superoxide radicals and H2O2) for 90 min or 50µM H2O2 for 1h, externalized PE and oxidative stress levels were increased in male hepatocytes to about the same extent for WT and KO mice. Female hepatocytes from WT and KO mice showed a similar increase with menadione but not with H2O2 treatment. Treatment of male or female hepatocytes with anti-CD95/FasL antibody for 6h caused a strong increase in caspase 3 activities in both WT and KO mice. Externalized PE levels were however not altered by apoptosis induction in both WT and KO cells.

Conclusions:

iPLA2ß deficiency increased basal oxidative stress and PE externalization in male but not female hepatocytes. Induced oxidative stress led to an increase in PE externalization in both male and female hepatocytes. PE externalization was correlated with oxidative stress but not apoptosis. Our results provide insights on the influence of iPLA2ß deficiency on oxidative stress-mediated PE externalization in a sex-dependent manner.