Knockdown of iPLA2γ enhances cisplatin-induced apoptosis by increasing ROS-dependent peroxidation of mitochondrial phospholipids in bladder cancer cells.

Cisplatin (CDDP) is a platinum-based drug with anti-cancer activity and is widely used as a standard therapy for bladder cancer. It is well known that CDDP causes cell death by increasing the generation of reactive oxygen species (ROS) and lipid peroxidation, but the mechanism of its anti-cancer effects has not been fully elucidated. There are still some problems such as chemoresistance in CDDP therapy. In the present study, we found the expression of Ca2+-independent phospholipase A2γ (iPLA2γ), which has been reported to regulate cellular redox homeostasis by inhibiting lipid peroxide accumulation, in human bladder cancer tissues. Thus, we investigated the effect of iPLA2γ knockdown on CDDP-induced bladder cancer cell death. As a result, we found that iPLA2γ knockdown significantly enhanced CDDP-induced apoptosis, intracellular and mitochondrial ROS production, cytochrome c release and caspase activation in bladder cancer cells. Moreover, mitochondrial membrane potential was decreased and peroxidation of mitochondrial phospholipids was increased by iPLA2γ knockdown. It was also shown that co-treatment of bromoenol lactone, an iPLA2 inhibitor, increased CDDP-induced apoptosis. These results indicated that iPLA2γ plays an important role in protecting bladder cancer cells from CDDP-induced apoptosis, and that iPLA2γ inhibitors might represent a novel strategy in CDDP-based multi-drug therapy.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

Differential Role of Liver X Receptor (LXR) α and LXRß in the Regulation of UDP-Glucuronosyltransferase 1A1 in Humanized UGT1 Mice.

Liver X receptors (LXRs), LXRα and LXRß, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 (hUGT1) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrß -/- , and hUGT1/Lxrαß -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXRα, with the induction pattern paralleling induction of LXRα-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαß -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXRα, showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXRα is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXRα, and not LXRß, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXRα is critical for the developmental expression of UGT1A1.

Long-chain acyl-CoA synthetase 4 participates in the formation of highly unsaturated fatty acid-containing phospholipids in murine macrophages.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

Calcium-independent phospholipase A2γ (iPLA2γ) and its roles in cellular functions and diseases.

Calcium-independent phospholipase A2γ (iPLA2γ)/patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is one of the iPLA2 enzymes, which do not require Ca2+ ion for their activity. iPLA2γ is a membrane-bound enzyme with unique features, including the utilization of four distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. This enzyme is preferentially distributed in the mitochondria and peroxisomes and is thought to be responsible for the maintenance of lipid homeostasis in these organelles. Thus, both the overexpression and the deletion of iPLA2γ in vivo caused mitochondrial abnormalities and dysfunction. Roles of iPLA2γ in lipid mediator production and cytoprotection against oxidative stress have also been suggested by in vitro and in vivo studies. The dysregulation of iPLA2γ can therefore be a critical factor in the development of many diseases, including metabolic diseases and cancer. In this review, we provide an overview of the biochemical properties of iPLA2γ and then summarize the current understanding of the in vivo roles of iPLA2γ revealed by knockout mouse studies.

Isothiocyanates induce UGT1A1 in humanized UGT1 mice in a CAR dependent fashion that is highly dependent upon oxidative stress.

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car-/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car-/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.