Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2γ-deficient mice

Group VIB Ca²⁺-independent phospholipase A₂γ (iPLA₂γ) is a membrane-bound iPLA₂ enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA₂γ by disrupting its gene in mice. iPLA₂γ-knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofilaments in iPLA₂γ-KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA₂γ-KO muscles. These results provide evidence that impairment of iPLA₂γ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA₂γ-KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA₂γ may contribute to modulation of lipid mediator production in vivo.     

Genetic Ablation of Calcium-independent Phospholipase A2�� Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

Genetic ablation of calcium-independent phospholipase A₂γ (iPLA₂γ) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA₂γ^−/− mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA₂γ in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA₂γ loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.     

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.     

Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry

The Group VIA-2 Ca²⁺-independent phospholipase A₂ (GVIA-2 iPLA₂) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA₂ in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA₂ as well as the interfacial lipid binding region.The Group VIA phospholipase A₂ is a member of the phospholipase A₂ superfamily that cleaves fatty acids from the sn-2 position of phospholipids (1, 2). The human Group VIA PLA₂³ gene yields multiple splice variants, including GVIA-1, GVIA-2, GVIA-3 PLA₂, GVIA Ankyrin-1, and GVIA Ankyrin-2 (3, 4). At least two isoforms, GVIA-1 and GVIA-2 iPLA₂, are active. Our laboratory purified and characterized the first mammalian iPLA₂, the 85-kDa GVIA-2 iPLA₂ (5), which became the first cloned iPLA₂ (6). This enzyme can hydrolyze the sn-2 fatty acyl bond of phospholipids and also has potent lysophospholipase and transacylase activity (7). GVIA iPLA₂ is involved in cell proliferation (8), apoptosis (9–11), bone formation (12), sperm development (13), and glucose-induced insulin secretion (14, 15), so its function may vary by cell and tissue.The human GVIA-2 iPLA₂ (806 amino acids), the form of the enzyme studied here, contains seven ankyrin repeats (residues 152–382), a linker region (residues 383–474) with the eighth repeat disrupted by a 54-amino acid insert (16), and a catalytic domain (residues 475–806). The active site serine of the GVIA iPLA₂ lies within a lipase consensus sequence (Gly-X-Ser⁵¹⁹-X-Gly) (1). The activity of GVIA iPLA₂ has been reported to be regulated through several mechanisms. A caspase-3 cleavage site at the N terminus of the enzyme has been identified that is clipped in vitro (17). This truncated form of the enzyme was hyperactive and reduced cell viability when overexpressed in HEK293 cells (17). Another possible control mechanism is through ATP binding on the ⁴⁸⁵GXGXXG motif (18).The activity of phospholipases depends critically on the interaction of the protein with phospholipid membranes. In vitro, GVIA iPLA₂ does not have any specificity for the fatty acid in the sn-2 position of substrate phospholipids (5). GVIA-2 iPLA₂ was found to be membrane-associated when overexpressed in COS-7 cells, and this was further confirmed in rat vascular smooth muscle cells (4, 19). The other active splice variant, GVIA-1, is cytosolic and not specific in targeting membrane surfaces (4, 19), indicating two different regulatory mechanisms between these two splice variants. The 54-residue insertion in the eighth ankyrin repeat alters the property of GVIA-2 iPLA₂ for membrane association. The ankyrin repeats have been reported to be involved in protein-protein interactions, such as 53BP2-p53, GA-binding protein α-GA-binding protein β, p16^INK4a-CDK6, and IκBα-NFκB (16). The ankyrin repeats of GVIA iPLA₂ may directly or indirectly assist membrane association because the catalytic domain by itself does not have activity (3). Determining the regions of the protein that interact with the membrane surface will allow for a more in-depth analysis of the regulatory mechanisms of the enzyme.There is an increasing interest in GVIA iPLA₂ because of its various newly discovered functions in vivo and in vitro. However, there is no published crystal or NMR structure to facilitate analysis on the molecular level. Amide hydrogen/deuterium exchange coupled with mass spectrometry (DXMS) has been widely used to analyze the interface of protein-protein interactions (20), protein conformational changes (21, 22), and protein dynamics (23), and we have now introduced it to study protein-phospholipid interactions (24, 25). There are also reports of using DXMS with homology modeling to validate enzymes where structural information does not exist (26). We used deuterium exchange along with homology modeling to generate models of the ankyrin repeats based on the Ankyrin-R (Protein Data Bank code 1N11) and of the catalytic domain based on patatin (Protein Data Bank code 1OXW). To study the interfacial activation of GVIA iPLA₂, we generated 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (PAPC) vesicles containing the methyl arachidonyl fluorophosphonate (MAFP) inhibitor, which binds to the active site and irreversibly inhibits GVIA iPLA₂ (7). By applying DXMS to the iPLA₂ and using our structural model, we were now able to monitor how GVIA iPLA₂ associates with phospholipid membranes.     

Calcium-independent phospholipase A2-catalyzed plasmalogen hydrolysis in hypoxic human coronary artery endothelial cells

Thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in activation of a membrane-associated, calcium-independent phospholipase A₂ (iPLA₂) that selectively hydrolyzes membrane plasmalogen phospholipids. Rupture of an atherosclerotic plaque and occlusion of the coronary vasculature results in a coronary ischemic event in which HCAEC in the ischemic area would be exposed to dramatic decreases in oxygen tension in addition to thrombin exposure. We exposed HCAEC to hypoxia in the presence or absence of thrombin stimulation and measured iPLA₂ activation, membrane phospholipid hydrolysis, and the accumulation of biologically active phospholipid metabolites. HCAEC exposed to hypoxia, thrombin stimulation, or a combination of the two conditions demonstrated an increase in iPLA₂ activity and an increase in arachidonic acid release from plasmenylcholine. Thrombin stimulation of normoxic HCAEC did not result in an accumulation of choline lysophospholipids, but hypoxia alone and in combination with thrombin stimulation led to a significant accumulation of lysoplasmenylcholine (LPlsCho). We propose that the presence of hypoxia inhibits LPlsCho catabolism, at least in part, as a result of the accumulation of long-chain acylcarnitines. The combination of increased production and decreased catabolism of LPlsCho is necessary for its accumulation. Pretreatment with bromoenol lactone to inhibit iPLA₂ blocked membrane phospholipid hydrolysis and production of membrane phospholipid-derived metabolites. The increase in iPLA₂ activity and the subsequent accumulation of membrane phospholipid-derived metabolites in HCAEC exposed to hypoxia or thrombin stimulation alone, and particularly in combination, have important implications in inflammation and arrhythmogenesis in atherosclerosis/thrombosis and subsequent myocardial ischemia. myocardial ischemia; arrhythmogenesis; thrombosis     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice

Calcium-independent phospholipase A₂β (iPLA₂β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA₂β-knockout (KO) mice. To clarify whether a deficiency of iPLA₂β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA₂β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA₂β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA₂β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA₂β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA₂β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA₂β-KO mice.     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.     

Activation of MAPKs in thrombin-stimulated ventricular myocytes is dependent on Ca2+-independent PLA2

Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca²⁺-independent PLA₂ (iPLA₂) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine. To determine whether MAPK regulates myocardial iPLA₂ activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated them with MAPK inhibitors before stimulating them with thrombin. Pretreatment with PD-98059 to inhibit p42/44 MAPK or SB-203580 to inhibit p38 MAPK had no significant effect on thrombin-stimulated, membrane-associated iPLA₂ activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAPK activity after 2 min. Pretreatment with the iPLA₂-selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAPK activity, suggesting that activation of MAPKs was dependent on iPLA₂ activation. Ventricular myocyte MAPK activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA₂-catalyzed membrane plasmalogen phospholipid hydrolysis. Altogether, these data suggest that activation of MAPKs occurs downstream of and is dependent on iPLA₂ activation in thrombin-stimulated rabbit ventricular myocytes. lysoplasmenylcholine; cell signaling; protease-activated receptors     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

The role of calcium-independent phospholipase A2 in cardiolipin remodeling in the spontaneously hypertensive heart failure rat heart

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)₄CL]. A decrease in (18:2)₄CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)₄CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A₂ (iPLA₂) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)₄CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA₂ inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)₄CL in the failing rat heart, and that mitochondrial iPLA₂ plays a role in the remodeling of CL''s acyl composition.     

Structure, Dynamics, and Ion Conductance of the Phospholamban Pentamer

A 52-residue membrane protein, phospholamban (PLN) is an inhibitor of an adenosine-5′-triphosphate-driven calcium pump, the Ca²⁺-ATPase. Although the inhibition of Ca²⁺-ATPase involves PLN monomers, in a lipid bilayer membrane, PLN monomers form stable pentamers of unknown biological function. The recent NMR structure of a PLN pentamer depicts cytoplasmic helices extending normal to the bilayer in what is known as the bellflower conformation. The structure shows transmembrane helices forming a hydrophobic pore 4 Å in diameter, which is reminiscent of earlier reports of possible ion conductance through PLN pentamers. However, recent FRET measurements suggested an alternative structure for the PLN pentamer, known as the pinwheel model, which features a narrower transmembrane pore and cytoplasmic helices that lie against the bilayer. Here, we report on structural dynamics and conductance properties of the PLN pentamers from all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Our AA simulations of the bellflower model demonstrate that in a lipid bilayer membrane or a detergent micelle, the cytoplasmic helices undergo large structural fluctuations, whereas the transmembrane pore shrinks and becomes asymmetric. Similar asymmetry of the transmembrane region was observed in the AA simulations of the pinwheel model; the cytoplasmic helices remained in contact with the bilayer. Using the CG approach, structural dynamics of both models were investigated on a microsecond timescale. The cytoplasmic helices of the CG bellflower model were observed to fall against the bilayer, whereas in the CG pinwheel model the conformation of the cytoplasmic helices remained stable. Using steered molecular dynamics simulations, we investigated the feasibility of ion conductance through the pore of the bellflower model. The resulting approximate potentials of mean force indicate that the PLN pentamer is unlikely to function as an ion channel.     

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A₂ (PLA₂). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7–10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A₂ (PLA₂). Indeed, higher levels of calcium-independent PLA₂ activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA₂ activity is associated with increases in the expression of the 80-kDa calcium-independent PLA₂ (iPLA₂). Together, these data suggest that the 80-kDa iPLA₂ may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.     

Ageing sensitized by iPLA2β deficiency induces liver fibrosis and intestinal atrophy involving suppression of homeostatic genes and alteration of intestinal lipids and bile acids

Ageing is a major risk factor for various forms of liver and gastrointestinal (GI) disease and genetic background may contribute to the pathogenesis of these diseases. Group VIA phospholipase A2 or iPLA₂β is a homeostatic PLA₂ by playing a role in phospholipid metabolism and remodeling. Global iPLA₂β^−/− mice exhibit aged-dependent phenotypes with body weight loss and abnormalities in the bone and brain. We have previously reported the abnormalities in these mutant mice showing susceptibility for chemical-induced liver injury and colitis. We hypothesize that iPLA₂β deficiency may sensitize with ageing for an induction of GI injury. Male wild-type and iPLA₂β^−/− mice at 4 and 20–22 months of age were studied. Aged, but not young, iPLA₂β^−/− mice showed increased hepatic fibrosis and biliary ductular expansion as well as severe intestinal atrophy associated with increased apoptosis, pro-inflammation, disrupted tight junction, and reduced number of mucin-containing globlet cells. This damage was associated with decreased expression of intestinal endoplasmic stress XBP1 and its regulator HNF1α, FATP4, ACSL5, bile-acid transport genes as well as nuclear receptors LXRα and FXR. By LC/MS-MS profiling, iPLA₂β deficiency in aged mice caused an increase of intestinal arachidonate-containing phospholipids concomitant with a decrease in ceramides. By the suppression of intestinal FXR/FGF-15 signaling, hepatic bile-acid synthesis gene expression was increased leading to an elevation of secondary and hydrophobic bile acids in liver, bile, and intestine. In conclusions, ageing sensitized by iPLA₂β deficiency caused a decline of key intestinal homeostatic genes resulting in the development of GI disease in a gut-to-liver manner.     

Evidence for proteolytic processing and stimulated organelle redistribution of iPLA2β

Over the past decade, important roles for the 84–88 kDa Group VIA Ca²⁺-independent phospholipase A₂ (iPLA₂β) in various organs have been described. We demonstrated that iPLA₂β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA₂β, and certain stimuli promote perinuclear localization of iPLA₂β. To gain a better understanding of its mobilization, iPLA₂β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA₂β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA₂β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA₂β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA₂β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA₂β in β-cells and we find here that these stimuli promote differential localization of iPLA₂β in subcellular organelles. Further, mass spectrometric analyses identified iPLA₂β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA₂β and redistribution of iPLA₂β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA₂β facilitates its participation in multiple biological processes.     

Role of Calcium Independent Phospholipase A<Subscript>2</Subscript> in Maintaining Mitochondrial Membrane Potential and Preventing Excessive Exocytosis in PC12 Cells

This study was carried out to elucidate the effects of calcium independent phospholipase A₂ (iPLA₂) on mitochondrial function and exocytosis in neuroendocrine cells. iPLA₂ mRNA and protein were detected in cell lysates and mitochondria from PC12 cells. Treatment of cells with the iPLA₂ inhibitor, bromoenol lactone (BEL), resulted in reduction in the mitochondrial membrane potential. Increase in membrane capacitance and number of spikes at amperometry, indicating exocytosis, were detected from PC12 cells after treatment with BEL. The induced exocytosis was abolished by pre-incubation of cells with the antioxidant, glutathione monoethyl ester, spin-trap/free radical scavenger, PBN, or inhibitors of the mitochondrial permeability transition pore, cyclosporine A and bongkrekic acid. These findings indicate that inhibition of iPLA₂ results in excessive exocytosis through increased oxidative damage (or failure to repair such damage) and defects in mitochondrial function. A similar process may occur in neurons with mutations in iPLA₂, leading to neuronal injury.