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Thematic Review Series:Phospholipases: Central Role in Lipid Signaling and Disease| Volume 56, ISSUE 9, P1643-1668, September 2015

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Calcium-independent phospholipases A2 and their roles in biological processes and diseases

Open AccessPublished:May 28, 2015DOI:https://doi.org/10.1194/jlr.R058701
      Among the family of phospholipases A2 (PLA2s) are the Ca2+-independent PLA2s (iPLA2s) and they are designated group VI iPLA2s. In relation to secretory and cytosolic PLA2s, the iPLA2s are more recently described and details of their expression and roles in biological functions are rapidly emerging. The iPLA2s or patatin-like phospholipases (PNPLAs) are intracellular enzymes that do not require Ca2+ for activity, and contain lipase (GXSXG) and nucleotide-binding (GXGXXG) consensus sequences. Though nine PNPLAs have been recognized, PNPLA8 (membrane-associated iPLA2γ) and PNPLA9 (cytosol-associated iPLA2β) are the most widely studied and understood. The iPLA2s manifest a variety of activities in addition to phospholipase, are ubiquitously expressed, and participate in a multitude of biological processes, including fat catabolism, cell differentiation, maintenance of mitochondrial integrity, phospholipid remodeling, cell proliferation, signal transduction, and cell death. As might be expected, increased or decreased expression of iPLA2s can have profound effects on the metabolic state, CNS function, cardiovascular performance, and cell survival; therefore, dysregulation of iPLA2s can be a critical factor in the development of many diseases. This review is aimed at providing a general framework of the current understanding of the iPLA2s and discussion of the potential mechanisms of action of the iPLA2s and related involved lipid mediators.

      OVERVIEW OF THE GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASES A2

      The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s that hydrolyze the sn-2 substituent from membrane phospholipids to release a free fatty acid and a lysolipid (
      • Dennis E.A.
      • Cao J.
      • Hsu Y.H.
      • Magrioti V.
      • Kokotos G.
      Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.
      ,
      • Gijón M.A.
      • Leslie C.C.
      Phospholipases A2.
      ). These enzymes are ubiquitously expressed, and in contrast to secretory PLA2s (sPLA2s) and cytosolic PLA2s (cPLA2s), do not require Ca2+ for either translocation or activity. Some of the first descriptions of iPLA2 activity were in the mid- to late-1980s with the identification of a plasmalogen-selective PLA2 in the cytosol of canine myocardium (
      • Wolf R.A.
      • Gross R.W.
      Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium.
      ) that migrated with a molecular mass of 40 kDa. Analogous activity was subsequently described in insulinoma cells (
      • Ramanadham S.
      • Wolf M.J.
      • Jett P.A.
      • Gross R.W.
      • Turk J.
      Characterization of an ATP-stimulatable Ca2+-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor.
      ) and renal proximal tubules (
      • Portilla D.
      • Shah S.V.
      • Lehman P.A.
      • Creer M.H.
      Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.
      ), as well as in a macrophage cell line (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      ). The Ca2+-independent PLA2s are designated as group VI iPLA2s (
      • Dennis E.A.
      The growing phospholipase A2 superfamily of signal transduction enzymes.
      ,
      • Balsinde J.
      • Dennis E.A.
      Function and inhibition of intracellular calcium-independent phospholipase A2.
      ) and now include seven members, as described in Table 1: iPLA2β (VIA-1 and -2), iPLA2γ (VIB), iPLA2δ (VIC), iPLA2ε (VID), iPLA2ζ (VIE), and iPLA2η (VIF). Three others (iPLA2φ, iPLA2ι, and iPLA2κ) have been recognized, but very little is known about them and they are not yet assigned to group VI (
      • Kienesberger P.C.
      • Oberer M.
      • Lass A.
      • Zechner R.
      Mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions.
      ,
      • Wilson P.A.
      • Gardner S.D.
      • Lambie N.M.
      • Commans S.A.
      • Crowther D.J.
      Characterization of the human patatin-like phospholipase family.
      ). Due to their shared homology with patatin, the iPLA2s are included in the patatin-like protein family and are also referred to as PNPLAs. The iPLA2s also share a consensus GXSXG catalytic motif contained within a patatin-like lipase domain. This review discusses the current understanding of the various iPLA2s, starting with the less-described iPLA2δ, iPLA2ε, iPLA2ζ, and iPLA2η, followed by emerging reports relating to iPLA2γ, and ending with the most widely examined, iPLA2β.
      TABLE 1Listing of the group VI family of iPLA2s
      Group VI PLA2sDescribedOther NamesChromosomeAmino AcidskDaAnk RepeatsLocalizationActive Site
      VIA-1, iPLA2β1994 (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      )
      PNPLA922q13.1752858CytosolS465
      VIA-2, iPLA2β1999 (
      • Ma Z.
      • Wang X.
      • Nowatzke W.
      • Ramanadham S.
      • Turk J.
      Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1.
      )
      PNPLA922q13.1804887CytosolS519
      VIB, iPLA2γ2000 (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2.
      ,
      • Tanaka H.
      • Takeya R.
      • Sumimoto H.
      A novel intracellular membrane-bound calcium-independent phospholipase A2.
      )
      PNPLA87q31782900MembraneS483
      VIC, iPLA2δ2002 (
      • van Tienhoven M.
      • Atkins J.
      • Li Y.
      • Glynn P.
      Human neuropathy target esterase catalyzes hydrolysis of membrane lipids.
      )
      PNPLA6, NTE19p13.3-13.21,3661460Neurons (ER, Golgi)S1005
      VID, iPLA2ε2001 (
      • Baulande S.
      • Lasnier F.
      • Lucas M.
      • Pairault J.
      Adiponutrin, a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage.
      )
      PNPLA3, adiponutrin22q13.31481520Liver adipocytesS47
      VIE, iPLA2ζ2004 (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ,
      • Villena J.A.
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.
      ,
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.
      )
      PNPLA2, desnutrin, ATGL11p15.5504550White and brown adipocytes (lipid droplets)S47
      VIF, iPLA2η1994 (
      • Lee W.C.
      • Salido E.
      • Yen P.H.
      Isolation of a new gene GS2 (DXS1283E) from a CpG island between STS and KAL1 on Xp22.3.
      )
      PNPLA4, gene sequence-2 (GS2)xp22.3253270ubiquitousS43

      iPLA2δ

      The group VIC iPLA2δ (PNPLA6), also known as neuropathy target esterase (NTE), was recognized for manifesting iPLA2 and lysophospholipase activities in 2002 (
      • van Tienhoven M.
      • Atkins J.
      • Li Y.
      • Glynn P.
      Human neuropathy target esterase catalyzes hydrolysis of membrane lipids.
      ). The gene for iPLA2δ is located at chromosome 19p13.3-13.2, and encodes a protein containing 1,366 amino acids with a molecular mass of 146 kDa and an active site at S1005. Expressed predominantly in neurons, iPLA2δ localizes to the endoplasmic reticulum (ER) and Golgi apparatus (
      • Baulande S.
      • Lasnier F.
      • Lucas M.
      • Pairault J.
      Adiponutrin, a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage.
      ), and its inhibition or deletion leads to axonal degeneration. It is in this context that NTE was discovered during studies for causes of organophosphorus ester-induced paralysis in the late 1960s (
      • Johnson M.K.
      A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds.
      ,
      • Johnson M.K.
      The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase.
      ). Whereas conventional KO of NTE is embryonic lethal (
      • Winrow C.J.
      • Hemming M.L.
      • Allen D.M.
      • Quistad G.B.
      • Casida J.E.
      • Barlow C.
      Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity.
      ), conditional KO of NTE in the CNS leads to neurodegeneration (
      • Akassoglou K.
      • Malester B.
      • Xu J.
      • Tessarollo L.
      • Rosenbluth J.
      • Chao M.V.
      Brain-specific deletion of neuropathy target esterase/swisscheese results in neurodegeneration.
      ,
      • Read D.J.
      • Li Y.
      • Chao M.V.
      • Cavanagh J.B.
      • Glynn P.
      Neuropathy target esterase is required for adult vertebrate axon maintenance.
      ), suggesting loss of function as a causative factor in the development of neurological diseases. However, mutations in the catalytic site of NTE lead to hereditary spastic paraplegia (
      • Rainier S.
      • Bui M.
      • Mark E.
      • Thomas D.
      • Tokarz D.
      • Ming L.
      • Delaney C.
      • Richardson R.J.
      • Albers J.W.
      • Matsunami N.
      • et al.
      Neuropathy target esterase gene mutations cause motor neuron disease.
      ,
      • Rainier S.
      • Albers J.W.
      • Dyck P.J.
      • Eldevik O.P.
      • Wilcock S.
      • Richardson R.J.
      • Fink J.K.
      Motor neuron disease due to neuropathy target esterase gene mutation: clinical features of the index families.
      ), a symptom of NTE-motor neuron disorder. Clinical manifestations of NTE-motor neuron disorder are only evident when mutations are carried by both alleles, suggesting that the neurodegeneration results from production of abnormal NTE, rather than due to reduction in NTE activity (
      • Hein N.D.
      • Stuckey J.A.
      • Rainier S.R.
      • Fink J.K.
      • Richardson R.J.
      Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties.
      ,
      • Hein N.D.
      • Rainier S.R.
      • Richardson R.J.
      • Fink J.K.
      Motor neuron disease due to neuropathy target esterase mutation: enzyme analysis of fibroblasts from human subjects yields insights into pathogenesis.
      ). Among the syndromes associated with mutations in PNPLA6 are: Gordon Holmes (
      • Topaloglu A.K.
      • Lomniczi A.
      • Kretzschmar D.
      • Dissen G.A.
      • Kotan L.D.
      • McArdle C.A.
      • Koc A.F.
      • Hamel B.C.
      • Guclu M.
      • Papatya E.D.
      • et al.
      Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.
      ,
      • Synofzik M.
      • Gonzalez M.A.
      • Lourenco C.M.
      • Coutelier M.
      • Haack T.B.
      • Rebelo A.
      • Hannequin D.
      • Strom T.M.
      • Prokisch H.
      • Kernstock C.
      • et al.
      PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.
      ) and Boucher-Neuhauser (
      • Synofzik M.
      • Gonzalez M.A.
      • Lourenco C.M.
      • Coutelier M.
      • Haack T.B.
      • Rebelo A.
      • Hannequin D.
      • Strom T.M.
      • Prokisch H.
      • Kernstock C.
      • et al.
      PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum.
      ), characterized by early-onset ataxia and hypogonadism; Oliver-McFarlane (
      • Hufnagel R.B.
      • Arno G.
      • Hein N.D.
      • Hersheson J.
      • Prasad M.
      • Anderson Y.
      • Krueger L.A.
      • Gregory L.C.
      • Stoetzel C.
      • Jaworek T.J.
      • et al.
      Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes.
      ), characterized by trichomegaly, congenital hypopituitarism, retinal degeneration, and choroidal atrophy; Laurence-Moon (
      • Hufnagel R.B.
      • Arno G.
      • Hein N.D.
      • Hersheson J.
      • Prasad M.
      • Anderson Y.
      • Krueger L.A.
      • Gregory L.C.
      • Stoetzel C.
      • Jaworek T.J.
      • et al.
      Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes.
      ), characterized by progressive spinocerebellar ataxia and spastic paraplegia; and photoreceptor degeneration and childhood blindness (
      • Kmoch S.
      • Majewski J.
      • Ramamurthy V.
      • Cao S.
      • Fahiminiya S.
      • Ren H.
      • MacDonald I.M.
      • Lopez I.
      • Sun V.
      • Keser V.
      • et al.
      Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.
      ). An NTE-related iPLA2 (PNPLA7) awaits further characterization (
      • Kienesberger P.C.
      • Oberer M.
      • Lass A.
      • Zechner R.
      Mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions.
      ,
      • Wilson P.A.
      • Gardner S.D.
      • Lambie N.M.
      • Commans S.A.
      • Crowther D.J.
      Characterization of the human patatin-like phospholipase family.
      ).

      iPLA2ε

      The group VID iPLA2ε (PNPLA3), also referred to as adiponutrin, was described in 2001 (
      • Baulande S.
      • Lasnier F.
      • Lucas M.
      • Pairault J.
      Adiponutrin, a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage.
      ). The gene for iPLA2ε is located at chromosome 22q13.31 and encodes a protein containing 481 amino acids with a molecular mass of 52 kDa and an active site at S47. PNPLA3 is mainly expressed in intracellular membrane fractions in hepatocytes (
      • He S.
      • McPhaul C.
      • Li J.Z.
      • Garuti R.
      • Kinch L.
      • Grishin N.V.
      • Cohen J.C.
      • Hobbs H.H.
      A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.
      ) and was originally described as a nutritionally-regulated adipose-specific transcript in 3T3-L1 adipocytes (
      • Baulande S.
      • Lasnier F.
      • Lucas M.
      • Pairault J.
      Adiponutrin, a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage.
      ). In addition to phospholipase activity, iPLA2ε manifests TG lipase and acylglycerol transacylase activities (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ), leading to the suggestion that it facilitates energy/lipid mobilization and storage in adipocytes. In this regard, iPLA2ε correlates highly with the development and progression of nonalcoholic fatty liver disease and has been identified as a genetic determinant of liver fibrosis (
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • Boerwinkle E.
      • Cohen J.C.
      • Hobbs H.H.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ,
      • Yuan X.
      • Waterworth D.
      • Perry J.R.
      • Lim N.
      • Song K.
      • Chambers J.C.
      • Zhang W.
      • Vollenweider P.
      • Stirnadel H.
      • Johnson T.
      • et al.
      Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.
      ). Whereas the WT PNPLA3 exhibits lipolytic activity toward TGs, the rs738409 variant PNPLA3, where isoleucine148 is replaced by methionine (L148M), reduces the access of substrates and activity of PNPLA3 toward glycerolipids. This leads to development of macrovesicular steatosis (
      • He S.
      • McPhaul C.
      • Li J.Z.
      • Garuti R.
      • Kinch L.
      • Grishin N.V.
      • Cohen J.C.
      • Hobbs H.H.
      A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.
      ,
      • Pirazzi C.
      • Adiels M.
      • Burza M.A.
      • Mancina R.M.
      • Levin M.
      • Stahlman M.
      • Taskinen M.R.
      • Orho-Melander M.
      • Perman J.
      • Pujia A.
      • et al.
      Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) affects hepatic VLDL secretion in humans and in vitro.
      ), simple steatosis to steatohepatitis and progressive cirrhosis (
      • Valenti L.
      • Alisi A.
      • Nobili V.
      I148M PNPLA3 variant and progressive liver disease: a new paradigm in hepatology.
      ), and hepatic fibrinogenesis by a sterol regulatory element-binding protein (SREBP)-1c-PNPLA3 pathway (
      • Krawczyk M.
      • Grunhage F.
      • Lammert F.
      Identification of combined genetic determinants of liver stiffness within the SREBP1c-PNPLA3 pathway.
      ). A greater impact of the L148M variant on hepatic lipid content is unmasked in the presence of other risk factors such as obesity (
      • Romeo S.
      • Sentinelli F.
      • Dash S.
      • Yeo G.S.
      • Savage D.B.
      • Leonetti F.
      • Capoccia D.
      • Incani M.
      • Maglio C.
      • Iacovino M.
      • et al.
      Morbid obesity exposes the association between PNPLA3 I148M (rs738409) and indices of hepatic injury in individuals of European descent.
      ), visceral adiposity (
      • Giudice E.M.
      • Grandone A.
      • Cirillo G.
      • Santoro N.
      • Amato A.
      • Brienza C.
      • Savarese P.
      • Marzuillo P.
      • Perrone L.
      The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat.
      ), increased intake of sugars (
      • Davis J.N.
      • Le K.A.
      • Walker R.W.
      • Vikman S.
      • Spruijt-Metz D.
      • Weigensberg M.J.
      • Allayee H.
      • Goran M.I.
      Increased hepatic fat in overweight Hispanic youth influenced by interaction between genetic variation in PNPLA3 and high dietary carbohydrate and sugar consumption.
      ), omega-6 PUFAs (
      • Santoro N.
      • Savoye M.
      • Kim G.
      • Marotto K.
      • Shaw M.M.
      • Pierpont B.
      • Caprio S.
      Hepatic fat accumulation is modulated by the interaction between the rs738409 variant in the PNPLA3 gene and the dietary omega6/omega3 PUFA intake.
      ), glucokinase regulatory protein gene variant (
      • Santoro N.
      • Zhang C.K.
      • Zhao H.
      • Pakstis A.J.
      • Kim G.
      • Kursawe R.
      • Dykas D.J.
      • Bale A.E.
      • Giannini C.
      • Pierpont B.
      • et al.
      Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents.
      ), chronic hepatitis B (
      • Viganò M.
      • Valenti L.
      • Lampertico P.
      • Facchetti F.
      • Motta B.M.
      • D'Ambrosio R.
      • Romagnoli S.
      • Dongiovanni P.
      • Donati B.
      • Fargion S.
      • et al.
      Patatin-like phospholipase domain-containing 3 I148M affects liver steatosis in patients with chronic hepatitis B.
      ), and hepatocellular carcinoma (
      • Friedrich K.
      • Wannhoff A.
      • Kattner S.
      • Brune M.
      • Hov J.R.
      • Weiss K.H.
      • Antoni C.
      • Dollinger M.
      • Neumann-Haefelin C.
      • Seufferlein T.
      • et al.
      PNPLA3 in end-stage liver disease: alcohol consumption, hepatocellular carcinoma development, and transplantation-free survival.
      ). In contrast to these reports, PNPLA3 has been reported to restore lipid homeostasis (
      • Chamoun Z.
      • Vacca F.
      • Parton R.G.
      • Gruenberg J.
      PNPLA3/adiponutrin functions in lipid droplet formation.
      ) by mediating acylation of lysophospholipids and hydrolyzing TGs in the liver in a direct manner or by regulation by cofactors (
      • Huang Y.
      • Cohen J.C.
      • Hobbs H.H.
      Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.
      ,
      • Kumari M.
      • Schoiswohl G.
      • Chitraju C.
      • Paar M.
      • Cornaciu I.
      • Rangrez A.Y.
      • Wongsiriroj N.
      • Nagy H.M.
      • Ivanova P.T.
      • Scott S.A.
      • et al.
      Adiponutrin functions as a nutritionally regulated lysophosphatidic acid acyltransferase.
      ).

      iPLA2ζ

      The group VIE iPLA2ζ (PNPLA2), also known as TST2.2, desnutrin, and adipose TG lipase (ATGL), was described in 2004 (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ,
      • Villena J.A.
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis.
      ,
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.
      ). The gene for iPLA2ζ is located at chromosome 11p15.5 and encodes a protein containing 504 amino acids with a molecular mass of 55 kDa and an active site at S47. Similar to iPLA2ε, iPLA2ζ also exhibits TG lipase and acylglycerol transacylase activities (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ). For optimal activity, ATGL requires the cofactor comparative gene identification-58 (CGI-58), which amplifies the hydrolase activity 20-fold (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.
      ). Mutations in CGI-58, as in Chanarin-Dorfman syndrome (
      • Yamaguchi T.
      • Osumi T.
      Chanarin-Dorfman syndrome: deficiency in CGI-58, a lipid droplet-bound coactivator of lipase.
      ), lead to TGs in various tissues and decreases in both CGI-58 and ATGL have been reported to exacerbate myocardial steatosis and oxidative stress to promote cardiac apoptosis in a rodent T2D model (
      • Inoue T.
      • Kobayashi K.
      • Inoguchi T.
      • Sonoda N.
      • Maeda Y.
      • Hirata E.
      • Fujimura Y.
      • Miura D.
      • Hirano K.
      • Takayanagi R.
      Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice.
      ). Analogously, ATGL deficiency in mice promotes tissue accumulation of lipids and leads to premature death due to cardiomyopathy, as a consequence of reductions in fatty acid oxidative gene expression, mitochondrial fatty acid oxidation, and reduced oxygen consumption (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.
      ). Macrophages with ATGL deficiency are more susceptible to ceramide-mediated mitochondrial dysfunction and programmed cell death (
      • Aflaki E.
      • Doddapattar P.
      • Radovic B.
      • Povoden S.
      • Kolb D.
      • Vujic N.
      • Wegscheider M.
      • Koefeler H.
      • Hornemann T.
      • Graier W.F.
      • et al.
      C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.
      ). β-Cell-specific ATGL-deficiency has been demonstrated to lead to hyperglycemia due to impaired insulin secretion, as a consequence of increased islet TG content with lower fatty acid levels. These mice also have decreased expression of PPARδ genes that encode enzymes required in mitochondrial oxidation, and this is reflected by impaired mitochondrial respiration and ATP production needed for glucose-stimulated insulin secretion (
      • Tang T.
      • Abbott M.J.
      • Ahmadian M.
      • Lopes A.B.
      • Wang Y.
      • Sul H.S.
      Desnutrin/ATGL activates PPARdelta to promote mitochondrial function for insulin secretion in islet beta cells.
      ). While polymorphisms in PNPLA2 are reported to highly correlate with T2D (
      • Schoenborn V.
      • Heid I.M.
      • Vollmert C.
      • Lingenhel A.
      • Adams T.D.
      • Hopkins P.N.
      • Illig T.
      • Zimmermann R.
      • Zechner R.
      • Hunt S.C.
      • et al.
      The ATGL gene is associated with free fatty acids, triglycerides, and type 2 diabetes.
      ), the contribution of ATGL to insulin secretion and signaling has been challenged (
      • Kienesberger P.C.
      • Lee D.
      • Pulinilkunnil T.
      • Brenner D.S.
      • Cai L.
      • Magnes C.
      • Koefeler H.C.
      • Streith I.E.
      • Rechberger G.N.
      • Haemmerle G.
      • et al.
      Adipose triglyceride lipase deficiency causes tissue-specific changes in insulin signaling.
      ,
      • Peyot M.L.
      • Guay C.
      • Latour M.G.
      • Lamontagne J.
      • Lussier R.
      • Pineda M.
      • Ruderman N.B.
      • Haemmerle G.
      • Zechner R.
      • Joly E.
      • et al.
      Adipose triglyceride lipase is implicated in fuel- and non-fuel-stimulated insulin secretion.
      ). In addition to its links to CGI-58 and PPARδ, ATGL has been reported to interact with TNFα in adipocytes (
      • Kim J.Y.
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3–L1 adipocytes and is a target for transactivation by PPARgamma.
      ), estrogen receptor α (ERα) in bone marrow (
      • Wend K.
      • Wend P.
      • Drew B.G.
      • Hevener A.L.
      • Miranda-Carboni G.A.
      • Krum S.A.
      ERα regulates lipid metabolism in bone through ATGL and perilipin.
      ), fat-specific protein 27 (FSP27) in human adipocytes (
      • Grahn T.H.
      • Kaur R.
      • Yin J.
      • Schweiger M.
      • Sharma V.M.
      • Lee M.J.
      • Ido Y.
      • Smas C.M.
      • Zechner R.
      • Lass A.
      • et al.
      Fat-specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) to regulate lipolysis and insulin sensitivity in human adipocytes.
      ), sirtuin 1 (SIRT1) during β-adrenergic signaling (
      • Khan S.A.
      • Sathyanarayan A.
      • Mashek M.T.
      • Ong K.T.
      • Wollaston-Hayden E.E.
      • Mashek D.G.
      ATGL-catalyzed lipolysis regulates SIRT1 to control PGC-1α/PPAR-α signaling.
      ), hepatic PPARα (
      • Ong K.T.
      • Mashek M.T.
      • Davidson N.O.
      • Mashek D.G.
      Hepatic ATGL mediates PPAR-alpha signaling and fatty acid channeling through an L-FABP independent mechanism.
      ), AMPK during thermogenesis (
      • Ahmadian M.
      • Abbott M.J.
      • Tang T.
      • Hudak C.S.
      • Kim Y.
      • Bruss M.
      • Hellerstein M.K.
      • Lee H.Y.
      • Samuel V.T.
      • Shulman G.I.
      • et al.
      Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype.
      ), and to be a candidate for transcriptional control by PPARγ-mediated signals (
      • Kim J.Y.
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3–L1 adipocytes and is a target for transactivation by PPARgamma.
      ).

      iPLA2η

      The group VIF iPLA2η (PNPLA4), also known as gene sequence-2 (GS2), was described in 1994 (
      • Lee W.C.
      • Salido E.
      • Yen P.H.
      Isolation of a new gene GS2 (DXS1283E) from a CpG island between STS and KAL1 on Xp22.3.
      ). The gene for iPLA2η is located at xp22.3 and encodes a protein containing 253 amino acids with a molecular mass of 27 kDa and an active site at S43. Similar to iPLA2ε and iPLA2ζ, iPLA2η exhibits TG lipase and acylglycerol transacylase activities (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ). Though expression of iPLA2η in a variety of tissues (liver, brain, skeletal muscle, lung, placenta, kidney, and pancreas) was identified in 1994, and more recently in adipose tissue (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ), to date, very little is known about its biology or its role in metabolic diseases. Similar to iPLA2ε and iPLA2ζ, iPLA2η activation is proposed to contribute to regulation of anabolic and catabolic fluxes of acyl equivalents in tissues. It has been suggested that the TG lipase activity of iPLA2ε, iPLA2ζ, and iPLA2η play roles in serum fatty acid accumulations associated with metabolic syndrome and T2D. A related GS2-like iPLA2 (PNPLA5) has yet to be characterized (
      • Kienesberger P.C.
      • Oberer M.
      • Lass A.
      • Zechner R.
      Mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions.
      ,
      • Wilson P.A.
      • Gardner S.D.
      • Lambie N.M.
      • Commans S.A.
      • Crowther D.J.
      Characterization of the human patatin-like phospholipase family.
      ).

      iPLA2γ

      The group VIB iPLA2γ (PNPLA8) genomic organization and mRNA sequence were first described in a variety of tissues (skeletal muscle, heart, placenta, brain, liver, and pancreas) in 2000 (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2.
      ) and later in the same year in lymphocytes (
      • Tanaka H.
      • Takeya R.
      • Sumimoto H.
      A novel intracellular membrane-bound calcium-independent phospholipase A2.
      ). The gene for iPLA2γ is located at 7q31 and encodes a protein containing 782 amino acids with a molecular mass of 90 kDa and an active site at S483. Recognition of the similarity in the catalytic domain between human iPLA2γ, cPLA2, and plant PLA2 patatin and conservation of sequence surrounding Asp627, and noting that substitution of alanine for either Ser483 of Asp627 caused loss of iPLA2γ activity, led to the suggestion that the Ser-Asp dyad constitutes the active site in human iPLA2γ (
      • Tanaka H.
      • Minakami R.
      • Kanaya H.
      • Sumimoto H.
      Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma).
      ). Initially recognized as membrane associated (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2.
      ,
      • Tanaka H.
      • Takeya R.
      • Sumimoto H.
      A novel intracellular membrane-bound calcium-independent phospholipase A2.
      ), dual-competing subcellular localization signals have been identified in discrete isoforms of iPLA2γ (
      • Mancuso D.J.
      • Jenkins C.M.
      • Sims H.F.
      • Cohen J.M.
      • Yang J.
      • Gross R.W.
      Complex transcriptional and translational regulation of iPLA2gamma resulting in multiple gene products containing dual competing sites for mitochondrial or peroxisomal localization.
      ) that promote its accumulation and expression of activity in the peroxisomes and mitochondria (
      • Mancuso D.J.
      • Han X.
      • Jenkins C.M.
      • Lehman J.J.
      • Sambandam N.
      • Sims H.F.
      • Yang J.
      • Yan W.
      • Yang K.
      • Green K.
      • et al.
      Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma.
      ), leading to the suggestion that iPLA2γ plays a role in integration of lipid and energy metabolism. Further, iPLA2 activity in the ER of rabbit and rat kidney (
      • Kinsey G.R.
      • Cummings B.S.
      • Beckett C.S.
      • Saavedra G.
      • Zhang W.
      • McHowat J.
      • Schnellmann R.G.
      Identification and distribution of endoplasmic reticulum iPLA2.
      ) and ventricular myocyte membranes (
      • Beckett C.S.
      • McHowat J.
      Calcium-independent phospholipase A2 in rabbit ventricular myocytes.
      ) has been demonstrated to be due to iPLA2γ.
      The iPLA2γ protein contains four methionine residues that can act as potential translational initiation sites (
      • Lee W.C.
      • Salido E.
      • Yen P.H.
      Isolation of a new gene GS2 (DXS1283E) from a CpG island between STS and KAL1 on Xp22.3.
      ,
      • Tanaka H.
      • Minakami R.
      • Kanaya H.
      • Sumimoto H.
      Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma).
      ) to generate the full-length (∼88 kDa) and three truncated products (77, 74, and 63 kDa). Attempts at expression of the truncated products in HEK293 cells, however, led to the predominant expression of the 63 kDa product (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • et al.
      Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.
      ), the isoform reported earlier to be expressed in peroxisomes (
      • Mancuso D.J.
      • Jenkins C.M.
      • Sims H.F.
      • Cohen J.M.
      • Yang J.
      • Gross R.W.
      Complex transcriptional and translational regulation of iPLA2gamma resulting in multiple gene products containing dual competing sites for mitochondrial or peroxisomal localization.
      ). Further examination of parental cells revealed that the 63 kDa isoform was much more abundant than the full-length iPLA2γ in HEK293 and human colorectal cancer cell lines, HCA-7 and WiDr, while in human bronchial epithelial (BEAS-2B) and rat fibroblastic (3YI) cells, the full-length iPLA2γ was the predominant isoform (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • et al.
      Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.
      ). These authors suggested that iPLA2γ potentiates arachidonic acid (AA) release from various subclasses of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) to increase prostaglandin E2 (PGE2) production via cyclooxygenase (COX)-1 and -2, and this contributes to cell growth and tumorigenesis. In contrast, comparative substrate preference studies revealed that unlike cPLA2, which generates predominantly 1-palmitoyl lysophosphatidylcholine (LPC) and AA from 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine hydrolysis, and iPLA2β, which exhibits mixed PLA1/PLA2 activities and generates 1-palmitoyl LPC at an initial 3-fold rate greater than 2-arachidonoyl LPC, iPLA2γ overexpressed in and purified from Sf9 cells hydrolyzed saturated and monounsaturated fatty acids at equal rates from the sn-1 or sn-2 position in diacyl PC substrates. However, it was less effective in releasing PUFAs from the sn-2 position, as reflected by generation of 2-arachidonoyl LPC at a 10-fold faster rate than 1-palmitoyl LPC (
      • Yan W.
      • Jenkins C.M.
      • Han X.
      • Mancuso D.J.
      • Sims H.F.
      • Yang K.
      • Gross R.W.
      The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2gamma: identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling.
      ).
      Understanding the role of iPLA2γ-derived lipid signals has substantially advanced following the generation of mice with tissue-specific overexpression or global KO of iPLA2γ. Cardiac-specific overexpression of iPLA2γ presented multiple phenotypes that included reductions in myocardial phospholipid mass in fasted and fed states, accu­mu­lation of TGs with caloric restriction, acute fasting-induced hemodynamic dysfunction (that was accompanied by loosely packed and disorganized mitochondrial cristae), and elevated levels of 2-arachidonoyl-sn-glycero-3-phosphocholine and 2-docosahexaenoyl-sn-glycero-3-phosphocholine (
      • Mancuso D.J.
      • Han X.
      • Jenkins C.M.
      • Lehman J.J.
      • Sambandam N.
      • Sims H.F.
      • Yang J.
      • Yan W.
      • Yang K.
      • Green K.
      • et al.
      Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma.
      ). These findings were associated with increased expression not of the full-length, but of the 70 and 63 kDa iPLA2γ isoforms. Impairment in mitochondrial function is also evidenced in iPLA2γ-null mice, which exhibit growth retardation, cold intolerance, and increased mortality due to aortic stress that were associated with decreased myocardial function and O2 consumption (
      • Mancuso D.J.
      • Sims H.F.
      • Han X.
      • Jenkins C.M.
      • Guan S.P.
      • Yang K.
      • Moon S.H.
      • Pietka T.
      • Abumrad N.A.
      • Schlesinger P.H.
      • et al.
      Genetic ablation of calcium-independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.
      ). The iPLA2γ-null mice also become resistant to Western diet-induced increases in body weight, adiposity, circulating levels of cholesterol, glucose, and insulin; and insulin resistance, and glucose intolerance (
      • Song H.
      • Wohltmann M.
      • Bao S.
      • Ladenson J.H.
      • Semenkovich C.F.
      • Turk J.
      Mice deficient in group VIB phospholipase A2 (iPLA2gamma) exhibit relative resistance to obesity and metabolic abnormalities induced by a Western diet.
      ). Ability to utilize fat and carbohydrates is also affected in these mice in association with severe impairment in skeletal muscle mitochondrial oxidation of fatty acids. Subsequent studies revealed that marked decreases in cardiolipin molecular species containing 22:6 were an underlying cause for the mitochondrial uncoupling evident in the iPLA2γ-null mice (
      • Mancuso D.J.
      • Sims H.F.
      • Yang K.
      • Kiebish M.A.
      • Su X.
      • Jenkins C.M.
      • Guan S.
      • Moon S.H.
      • Pietka T.
      • Nassir F.
      • et al.
      Genetic ablation of calcium-independent phospholipase A2gamma prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation.
      ). Similarly, hippocampal phospholipid metabolism was found to be severely compromised with iPLA2γ-deficiency leading to a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction, that was associated with alterations in the compositions and content of PCs, PEs, oxidized PEs, and ceramides and a shift in cardiolipins to shorter chain molecular species (
      • Mancuso D.J.
      • Kotzbauer P.
      • Wozniak D.F.
      • Sims H.F.
      • Jenkins C.M.
      • Guan S.
      • Han X.
      • Yang K.
      • Sun G.
      • Malik I.
      • et al.
      Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction.
      ). Increased lipid peroxidation was also evident in the skeletal muscles of iPLA2γ-null mice, which exhibited abnormal mitochondrial function, oxidative stress, growth retardation, and loss of skeletal muscle structure and function (
      • Yoda E.
      • Hachisu K.
      • Taketomi Y.
      • Yoshida K.
      • Nakamura M.
      • Ikeda K.
      • Taguchi R.
      • Nakatani Y.
      • Kuwata H.
      • Murakami M.
      • et al.
      Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2gamma-deficient mice.
      ). These findings are consistent with a previous report of the protective effects of iPLA2γ, deduced using a selective inhibitor of iPLA2γ against oxidant-induced lipid peroxidation and necrosis of renal proximal tubular cells (
      • Kinsey G.R.
      • McHowat J.
      • Beckett C.S.
      • Schnellmann R.G.
      Identification of calcium-independent phospholipase A2gamma in mitochondria and its role in mitochondrial oxidative stress.
      ). In contrast to the earlier report in the heart (
      • Mancuso D.J.
      • Han X.
      • Jenkins C.M.
      • Lehman J.J.
      • Sambandam N.
      • Sims H.F.
      • Yang J.
      • Yan W.
      • Yang K.
      • Green K.
      • et al.
      Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma.
      ), the full-length iPLA2γ was the major isoform detected in the last two studies (
      • Yoda E.
      • Hachisu K.
      • Taketomi Y.
      • Yoshida K.
      • Nakamura M.
      • Ikeda K.
      • Taguchi R.
      • Nakatani Y.
      • Kuwata H.
      • Murakami M.
      • et al.
      Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2gamma-deficient mice.
      ,
      • Kinsey G.R.
      • McHowat J.
      • Beckett C.S.
      • Schnellmann R.G.
      Identification of calcium-independent phospholipase A2gamma in mitochondria and its role in mitochondrial oxidative stress.
      ).
      The identification of iPLA2γ in the mitochondria and ER paved the way for studies that demonstrated a protective role for iPLA2γ against cell death. Utilizing knockdown protocols, global iPLA2γ-null mouse model, or by selective inhibition with bromoenol lactone (BEL) (suicide substrate) of iPLA2γ (R-BEL) versus iPLA2β (S-BEL), several studies reported protective effects of iPLA2γ against oxidant- and cytokine-induced cell death. Human astrocytes exposed to hydrogen peroxide or tert-butyl hydroperoxide exhibited cell death, and pretreatment with S-BEL, but not R-BEL, amplified loss of ATP levels and cell necrosis (
      • Peterson B.
      • Knotts T.
      • Cummings B.S.
      Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death.
      ). Similarly, knockdown of iPLA2γ in renal proximal tubular cells resulted in increased susceptibility to oxidant-induced cell death, elevations in lipid peroxidation, and uncoupled oxygen consumption (
      • Kinsey G.R.
      • Blum J.L.
      • Covington M.D.
      • Cummings B.S.
      • McHowat J.
      • Schnellmann R.G.
      Decreased iPLA2gamma expression induces lipid peroxidation and cell death and sensitizes cells to oxidant-induced apoptosis.
      ,
      • Cummings B.S.
      • Gelasco A.K.
      • Kinsey G.R.
      • McHowat J.
      • Schnellmann R.G.
      Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.
      ). Analogous findings were reported in INS-1 insulinoma cells, where knockdown of iPLA2γ promoted increases in cytokine- and oxidant-induced membrane peroxidation and apoptosis (
      • Bao S.
      • Song H.
      • Tan M.
      • Wohltmann M.
      • Ladenson J.H.
      • Turk J.
      Group VIB phospholipase A2 promotes proliferation of INS-1 insulinoma cells and attenuates lipid peroxidation and apoptosis induced by inflammatory cytokines and oxidant agents.
      ). Cytoprotective effects of iPLA2γ were also demonstrated in glomerular epithelial cells, and these were attributed to iPLA2γ-mediated upregulation of ER chaperones (
      • Elimam H.
      • Papillon J.
      • Takano T.
      • Cybulsky A.V.
      Calcium-independent phospholipase A2gamma enhances activation of the ATF6 transcription factor during endoplasmic reticulum stress.
      ). A general conclusion derived from these studies is that the lack of iPLA2γ decreases substrate availability for reacylation, leading to increases in lipid peroxidation. In apparent contrast to these reports, genetic ablation of iPLA2γ or its inhibition with R-BEL attenuated calcium-, reactive oxygen species (ROS)-, or oxidized lipid-mediated increases in liver mitochondrial swelling, mitochondrial permeability transition pore opening, and cytochrome c release from mitochondria, which trigger the intrinsic apoptotic pathway (
      • Moon S.H.
      • Jenkins C.M.
      • Kiebish M.A.
      • Sims H.F.
      • Mancuso D.J.
      • Gross R.W.
      Genetic ablation of calcium-independent phospholipase A2gamma (iPLA2gamma) attenuates calcium-induced opening of the mitochondrial permeability transition pore and resultant cytochrome c release.
      ,
      • Brustovetsky T.
      • Antonsson B.
      • Jemmerson R.
      • Dubinsky J.M.
      • Brustovetsky N.
      Activation of calcium-independent phospholipase A2 (iPLA2) in brain mitochondria and release of apoptogenic factors by BAX and truncated BID.
      ).
      The more recent description of iPLA2γ, to date, has limited wide studies of its role in clinical diseases, but a few reports suggest a role for iPLA2γ in certain clinical-related disorders. Chagas' disease is caused by protozoan parasite Trypanosoma cruzi, which infects cardiac myocytes promoting release of inflammatory mediators such as eicosanoids. Inhibition of iPLA2γ attenuated AA and PGE2 release and platelet-activating factor (PAF) production from HL-1 cardiac myocytes infected with T. cruzi (
      • Sharma J.
      • Eickhoff C.S.
      • Hoft D.F.
      • Ford D.A.
      • Gross R.W.
      • McHowat J.
      The absence of myocardial calcium-independent phospholipase A2gamma results in impaired prostaglandin E2 production and decreased survival in mice with acute Trypanosoma cruzi infection.
      ), and these effects were alleviated by pretreatment with R-BEL. Consistent with a protective role of iPLA2γ in this process, the survival rates were lowered and tissue parasitism amplified in T. cruzi-infected iPLA2γ-null mice, suggesting that iPLA2γ activity affords protection against acute state cardiomyopathy in Chagas' disease (
      • Sharma J.
      • Eickhoff C.S.
      • Hoft D.F.
      • Ford D.A.
      • Gross R.W.
      • McHowat J.
      The absence of myocardial calcium-independent phospholipase A2gamma results in impaired prostaglandin E2 production and decreased survival in mice with acute Trypanosoma cruzi infection.
      ). In contrast, iPLA2γ-deficiency has been shown to increase bleeding time and provide resistance to thromboembolism (
      • Yoda E.
      • Rai K.
      • Ogawa M.
      • Takakura Y.
      • Kuwata H.
      • Suzuki H.
      • Nakatani Y.
      • Murakami M.
      • Hara S.
      Group VIB calcium-independent phospholipase A2 (iPLA2gamma) regulates platelet activation, hemostasis and thrombosis in mice.
      ), raising the possibility of targeting iPLA2γ for antithrombotic drug development. To date, the only reported clinical manifestation relating to iPLA2γ is a report that its absence is associated with myocardial dysfunction, cognitive defects, and mitochondrial degeneration (
      • Saunders C.J.
      • Moon S.H.
      • Liu X.
      • Thiffault I.
      • Coffman K.
      • LePichon J.B.
      • Taboada E.
      • Smith L.D.
      • Farrow E.G.
      • Miller N.
      • et al.
      Loss of function variants in human PNPLA8 encoding calcium-independent phospholipase A2 gamma recapitulate the mitochondriopathy of the homologous null mouse.
      ) in a case study that closely parallels the phenotype in iPLA2γ-null mice (
      • Mancuso D.J.
      • Kotzbauer P.
      • Wozniak D.F.
      • Sims H.F.
      • Jenkins C.M.
      • Guan S.
      • Han X.
      • Yang K.
      • Sun G.
      • Malik I.
      • et al.
      Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction.
      ).

      iPLA2β

      The group VIA iPLA2β (PNPLA9) is the most widely described of the iPLA2s and expression of its activity was first described in P388D1 macrophage-like cells in 1994 (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      ) and later shown to be the same enzyme (
      • Balboa M.A.
      • Balsinde J.
      • Jones S.S.
      • Dennis E.A.
      Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.
      ) as that cloned from Chinese hamster ovary cells in 1997 (
      • Balboa M.A.
      • Balsinde J.
      • Jones S.S.
      • Dennis E.A.
      Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.
      ,
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ,
      • Jones S.S.
      • Tang J.
      • Kriz R.
      • Shaffer M.
      • Knopf J.
      • Seehra J.
      Isolation, molecular cloning and expression of a novel calcium-independent phospholipase A2..
      ). Unlike cPLA2, which exhibits preference for hydrolysis of AA from the sn-2 position (
      • Ghosh M.
      • Tucker D.E.
      • Burchett S.A.
      • Leslie C.C.
      Properties of the group IV phospholipase A2 family.
      ), the iPLA2s demonstrate no substrate specificity and manifest PLA2/PLA1, lysophospholipase (
      • Wolf M.J.
      • Gross R.W.
      Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2.
      ,
      • Lio Y.C.
      • Dennis E.A.
      Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2.
      ), transacylase (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ,
      • Lio Y.C.
      • Dennis E.A.
      Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2.
      ), and thioesterase (
      • Jenkins C.M.
      • Yan W.
      • Mancuso D.J.
      • Gross R.W.
      Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2beta. Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity.
      ,
      • Carper M.J.
      • Zhang S.
      • Turk J.
      • Ramanadham S.
      Skeletal muscle group VIA phospholipase A2 (iPLA2β): expression and role in fatty acid oxidation.
      ) activities. The extensively studied iPLA2β was cloned from hamster (
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ), mouse (
      • Balboa M.A.
      • Balsinde J.
      • Jones S.S.
      • Dennis E.A.
      Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.
      ), and rat (
      • Ma Z.
      • Ramanadham S.
      • Kempe K.
      • Chi X.S.
      • Ladenson J.
      • Turk J.
      Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin.
      ), and they represent species homologs that are 85 kDa proteins (752 amino acids) with a serine lipase consensus sequence (GTSGT), preceded by eight N-terminal ankyrin (Ank) repeats (
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ,
      • Ma Z.
      • Ramanadham S.
      • Kempe K.
      • Chi X.S.
      • Ladenson J.
      • Turk J.
      Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin.
      ).
      A homologous 88 kDa iPLA2β was cloned from human lymphocyte lines and testis (
      • Larsson P.K.A.
      • Claesson H-E.
      • Kennedy B.P.
      Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity.
      ) that contains a 54-amino acid insert interrupting the eighth Ank repeat. Subsequent analyses with human pancreatic islet mRNA identified cDNA species that encoded two distinct 85 kDa (VIA-1) and 88 kDa (VIA-2) human iPLA2β isoforms (
      • Ma Z.
      • Wang X.
      • Nowatzke W.
      • Ramanadham S.
      • Turk J.
      Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1.
      ). Analogous transcripts were also identified in human promonocytic U937 cells. The human iPLA2β gene resides on chromosome 22 in region q13.1 and contains 16 exons in the VIA-2 transcript. Exon 8 is not present in the VIA-1 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing.
      Additional alternate splicing events generate iPLA2β variants that differ in their subcellular localization, catalytic activity, and likely cellular function (
      • Larsson P.K.A.
      • Claesson H-E.
      • Kennedy B.P.
      Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity.
      ). Splice variants (Ank-1 and Ank-2) encode premature stop codons due to alternatively spliced exon 10a. The proteins encoded by these splice variants, VIA Ank-1 (53 kDa, ∼479 amino acids) and VIA Ank-2 (47 kDa, ∼427 amino acids), terminate after the seventh Ank repeat domain and before the active site, whereas VIA-3 (∼70 kDa, 640 amino acids) terminates just after the lipase active site. Two additional active iPLA2β isoforms have been recognized to arise from proteolytic cleavage: a ∼63 kDa isoform (VIA-4, 623 amino acids) arising from caspase-3-catalyzed cleavage at the N terminal (
      • Atsumi G.
      • Tajima M.
      • Hadano A.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not cytosolic phospholipase A2, which undergoes proteolytic inactivation.
      ,
      • Ramanadham S.
      • Hsu F.F.
      • Zhang S.
      • Jin C.
      • Bohrer A.
      • Song H.
      • Bao S.
      • Ma Z.
      • Turk J.
      Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2β) and suppressed by inhibition of iPLA2β.
      ) and a ∼70 kDa (VIA-5, ∼640 amino acids) isoform arising from C-terminal cleavage (
      • Ramanadham S.
      • Song H.
      • Hsu F.F.
      • Zhang S.
      • Crankshaw M.
      • Grant G.A.
      • Newgard C.B.
      • Bao S.
      • Ma Z.
      • Turk J.
      Pancreatic islets and insulinoma cells express a novel isoform of group VIA phospholipase A2 (iPLA2β) that participates in glucose-stimulated insulin secretion and is not produced by alternate splicing of the iPLA2β transcript.
      ). Proteomic analyses by mass spectrometry further reveal that the iPLA2β is a candidate for numerous truncations at the N-terminal end (
      • Song H.
      • Bao S.
      • Lei X.
      • Jin C.
      • Zhang S.
      • Turk J.
      • Ramanadham S.
      Evidence for proteolytic processing and stimulated organelle redistribution of iPLA2β.
      ,
      • Song H.
      • Hecimovic S.
      • Goate A.
      • Hsu F.F.
      • Bao S.
      • Vidavsky I.
      • Ramanadham S.
      • Turk J.
      Characterization of N-terminal processing of group VIA phospholipase A2 and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests.
      ), but the activities and biological roles manifested by these products have not yet been discerned.

      Basic characteristics of iPLA2β

      In addition to the Ank repeats, the iPLA2β protein contains an ATP binding consensus motif (GGGVKG), an N-terminal caspase-3 cleavage site (DVTD), and a putative bipartite nuclear localization sequence (KREFGEHTKMTDVKKPK). Though under basal conditions iPLA2β is predominantly localized in the cytosol (
      • Gross R.W.
      • Ramanadham S.
      • Kruszka K.K.
      • Han X.
      • Turk J.
      Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells.
      ), translocation of iPLA2β to the Golgi, ER, mitochondria, and nucleus is evident under stimulatory conditions (
      • Ramanadham S.
      • Hsu F.F.
      • Zhang S.
      • Jin C.
      • Bohrer A.
      • Song H.
      • Bao S.
      • Ma Z.
      • Turk J.
      Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2β) and suppressed by inhibition of iPLA2β.
      ,
      • Song H.
      • Bao S.
      • Lei X.
      • Jin C.
      • Zhang S.
      • Turk J.
      • Ramanadham S.
      Evidence for proteolytic processing and stimulated organelle redistribution of iPLA2β.
      ,
      • Lei X.
      • Zhang S.
      • Bohrer A.
      • Bao S.
      • Song H.
      • Ramanadham S.
      The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase.
      ,
      • Lei X.
      • Zhang S.
      • Bohrer A.
      • Ramanadham S.
      Calcium-independent phospholipase A2 (iPLA2β)-mediated ceramide generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis.
      ,
      • Ma Z.
      • Ramanadham S.
      • Wohltmann M.
      • Bohrer A.
      • Hsu F.F.
      • Turk J.
      Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2β) indicate a signaling rather than a housekeeping role for iPLA2β.
      ,
      • Turk J.
      • Ramanadham S.
      The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2β) in beta-cells.
      ,
      • Ma Z.
      • Zhang S.
      • Turk J.
      • Ramanadham S.
      Stimulation of insulin secretion and associated nuclear accumulation of iPLA2β in INS-1 insulinoma cells.
      ,
      • Shinzawa K.
      • Tsujimoto Y.
      PLA2 activity is required for nuclear shrinkage in caspase-independent cell death.
      ,
      • Bao S.
      • Jin C.
      • Zhang S.
      • Turk J.
      • Ma Z.
      • Ramanadham S.
      Beta-cell calcium-independent group VIA phospholipase A2 (iPLA2β): tracking iPLA2β movements in response to stimulation with insulin secretagogues in INS-1 cells.
      ,
      • Ramanadham S.
      • Song H.
      • Bao S.
      • Hsu F-F.
      • Zhang S.
      • Ma Z.
      • Jin C.
      • Turk J.
      Islet complex lipids: Involvement in the actions of group VIA calcium-independent phospholipase A2 in β-cells.
      ). The iPLA2β and iPLA2γ share signature ATP binding motif, serine lipase site, and a region of 9 amino acids (627-635 in iPLA2γ), whose biological significance is not known, but otherwise lack any additional homology (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2.
      ,
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ).

      Modulation of iPLA2β

      Oligomerization.

      A unique distinction between iPLA2β and other PLA2s is the presence of a variable number of Ank repeats in iPLA2β, which are absent in other PLA2s. Several lines of study suggest that the Ank regions confer iPLA2β protein activity. The active form of iPLA2β appears to be an oligomer of interacting protein subunits, as supported by radiation inactivation and gel filtration chromatography analyses that reveal association of the 85 kDa iPLA2β activity with an apparent molecular mass of 250–350 kDa (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      ,
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ,
      • Ramanadham S.
      • Wolf M.J.
      • Ma Z.
      • Li B.
      • Wang J.
      • Gross R.W.
      • Turk J.
      Evidence for association of an ATP-stimulatable Ca2+-independent phospholipase A2 from pancreatic islets and HIT insulinoma cells with a phosphofructokinase-like protein.
      ). This has led to the speculation that the active form of iPLA2β is an oligomer of 85 kDa subunits and that the subunits associate with each other via their Ank repeat regions (
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ), similar to the involvement of Ank repeats in other protein-protein interactions (
      • Bennett V.
      Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm.
      ). Consistent with this possibility are the observations that iPLA2β deletion mutants lacking the Ank repeat domain, but retaining the catalytic domain, are catalytically inactive (
      • Tang J.
      • Kriz R.W.
      • Wolfman N.
      • Shaffer M.
      • Seehra J.
      • Jones S.S.
      A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
      ) and that activity of the full-length protein is reduced when it is coexpressed with truncated iPLA2β-like proteins that retain the Ank repeat domain, but lack the catalytic domain (
      • Larsson P.K.A.
      • Claesson H-E.
      • Kennedy B.P.
      Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity.
      ). In the long isoform of human iPLA2, a proline-rich insert interrupts the last iPLA2β Ank repeat with some similarities to the Smad4 domain that mediates interactions with signaling partners (
      • de Caestecker M.P.
      • Hemmati P.
      • Larisch-Bloch S.
      • Ajmera R.
      • Roberts A.B.
      • Lechleider R.J.
      Characterization of functional domains within Smad4/DPC4.
      ). This raises the possibility that the proline-rich insert in human iPLA2β allows it to interact with proteins not recognized by the short isoform of iPLA2β.

      Oxidation.

      It has been suggested that iPLA2β inactivation can occur by a mechanism involving oxidation of sulfhydryl groups within the iPLA2β (
      • Cummings B.S.
      • Gelasco A.K.
      • Kinsey G.R.
      • McHowat J.
      • Schnellmann R.G.
      Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.
      ). Subsequently, oligomerization of iPLA2β in INS-1 cells in response to oxidative stress was demonstrated (
      • Song H.
      • Bao S.
      • Ramanadham S.
      • Turk J.
      Effects of biological oxidants on the catalytic activity and structure of group VIA phospholipase A2.
      ). Oxidant-induced oligomerization alters the subcellular localization of iPLA2β and results in reduced release of AA, suggesting inhibition of iPLA2β catalytic activity. These nonproductive oligomers are DTT-sensitive and therefore likely generated through intermolecular disulfide bonds. Like iPLA2β, iPLA2γ activity is suppressed by oxidants, but restored when oxidant-inhibited enzyme is treated with a reducing agent (
      • Cummings B.S.
      • Gelasco A.K.
      • Kinsey G.R.
      • McHowat J.
      • Schnellmann R.G.
      Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.
      ,
      • McHowat J.
      • Swift L.M.
      • Arutunyan A.
      • Sarvazyan N.
      Clinical concentrations of doxorubicin inhibit activity of myocardial membrane-associated, calcium-independent phospholipase A2.
      ).
      Together, these studies indicate that iPLA2β monomers are capable of assembling into both productive and nonproductive oligomers. The productive oligomerization is mediated through the N-terminal Ank repeat domain, while inactive oligomers are formed through intramolecular disulfide bonds.

      Activation.

      The iPLA2β protein contains a consensus nucleotide-binding motif (GGGVKG) that is homologous to those of protein kinases (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A2.
      ,
      • Ma Z.
      • Turk J.
      The molecular biology of the group VIA Ca2+-independent phospholipase A2.
      ). This feature mediates regulation and stabilization of iPLA2β activity by ATP (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      ,
      • Lio Y.C.
      • Dennis E.A.
      Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2.
      ,
      • Hazen S.L.
      • Gross R.W.
      ATP-dependent regulation of rabbit myocardial cytosolic calcium- independent phospholipase A2.
      ,
      • Hazen S.L.
      • Ford D.A.
      • Gross R.W.
      Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate.
      ), which is independent of enzyme phosphorylation (
      • Ackermann E.J.
      • Kempner E.S.
      • Dennis E.A.
      Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.
      ,
      • Hazen S.L.
      • Gross R.W.
      ATP-dependent regulation of rabbit myocardial cytosolic calcium- independent phospholipase A2.
      ,
      • Hazen S.L.
      • Ford D.A.
      • Gross R.W.
      Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate.
      ). Also contained in iPLA2β are a C-terminal 1-9-14 calmodulin-binding motif (IRKGQGNKVKK LSI) and a calmodulin-binding peptide (AWSEMVGIQYFR) (
      • Turk J.
      • Ramanadham S.
      The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2β) in beta-cells.
      ,
      • Ma Z.
      • Turk J.
      The molecular biology of the group VIA Ca2+-independent phospholipase A2.
      ,
      • Jenkins C.M.
      • Wolf M.J.
      • Mancuso D.J.
      • Gross R.W.
      Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A2beta. Implications for structure and function.
      ). These facilitate formation of a signaling complex between iPLA2β and CaMKIIβ and enhancement of both activities is evident upon their association (
      • Wang Z.
      • Ramanadham S.
      • Ma Z.A.
      • Bao S.
      • Mancuso D.J.
      • Gross R.W.
      • Turk J.
      Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells.
      ). This has been offered as one explanation of why Ca2+ store depletion activates iPLA2 (
      • Wolf M.J.
      • Wang J.
      • Turk J.
      • Gross R.W.
      Depletion of intracellular calcium stores activates smooth muscle cell calcium-independent phospholipase A2. A novel mechanism underlying arachidonic acid mobilization.
      ) and may occur in vascular myocytes (
      • Wolf M.J.
      • Gross R.W.
      Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2.
      ), pancreatic islet β-cells (
      • Nowatzke W.
      • Ramanadham S.
      • Ma Z.
      • Hsu F.F.
      • Bohrer A.
      • Turk J.
      Mass spectrometric evidence that agents that cause loss of Ca2+ from intracellular compartments induce hydrolysis of arachidonic acid from pancreatic islet membrane phospholipids by a mechanism that does not require a rise in cytosolic Ca2+ concentration.
      ), and human granulocytes (
      • Larsson Forsell P.K.
      • Runarsson G.
      • Ibrahim M.
      • Bjorkholm M.
      • Claesson H.E.
      On the expression of cytosolic calcium-independent phospholipase A2 (88 kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes.
      ). Ca2+ store depletion also activates hydrolysis of arachidonate from phospholipids in differentiated human U937 promonocytic cells by a mechanism that does not require a rise in cytosolic [Ca2+] (
      • Rzigalinski B.A.
      • Blackmore P.F.
      • Rosenthal M.D.
      Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells.
      ). The iPLA2β protein also contains a consensus sequence site for caspase-3-mediated cleavage within the first Ank repeat (
      • Atsumi G.
      • Murakami M.
      • Kojima K.
      • Hadano A.
      • Tajima M.
      • Kudo I.
      Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release.
      ). The truncated product manifests higher activity (
      • Atsumi G.
      • Murakami M.
      • Kojima K.
      • Hadano A.
      • Tajima M.
      • Kudo I.
      Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release.
      ) and localizes to the nucleus under high glucose stimulation and prolonged stress (
      • Ramanadham S.
      • Hsu F.F.
      • Zhang S.
      • Jin C.
      • Bohrer A.
      • Song H.
      • Bao S.
      • Ma Z.
      • Turk J.
      Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2β) and suppressed by inhibition of iPLA2β.
      ,
      • Ma Z.
      • Zhang S.
      • Turk J.
      • Ramanadham S.
      Stimulation of insulin secretion and associated nuclear accumulation of iPLA2β in INS-1 insulinoma cells.
      ), suggesting that it may amplify hydrolysis of nuclear membrane phospholipids and lead to nuclear membrane lysis, or that the iPLA2β and/or iPLA2β-derived products accumulate in an environment that can potentially participate in transcriptional induction of favorable and nonfavorable genes.

      Gene induction.

      The iPLA2β gene contains a sterol regulatory element (SRE) (
      • Seashols S.J.
      • del Castillo Olivares A.
      • Gil G.
      • Barbour S.E.
      Regulation of group VIA phospholipase A2 expression by sterol availability.
      ). Under stressful conditions SREBPs are processed to mature forms of SREBPs (
      • Akarte A.S.
      • Srinivasan B.P.
      • Gandhi S.
      Vildagliptin selectively ameliorates GLP-1, GLUT4, SREBP-1c mRNA levels and stimulates β-cell proliferation resulting in improved glucose homeostasis in rats with streptozotocin-induced diabetes.
      ,
      • Boslem E.
      • MacIntosh G.
      • Preston A.M.
      • Bartley C.
      • Busch A.K.
      • Fuller M.
      • Laybutt D.R.
      • Meikle P.J.
      • Biden T.J.
      A lipidomic screen of palmitate-treated MIN6 beta-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking.
      ,
      • Chan J.Y.
      • Cooney G.J.
      • Biden T.J.
      • Laybutt D.R.
      Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells.
      ,
      • Chin H.J.
      • Fu Y.Y.
      • Ahn J.M.
      • Na K.Y.
      • Kim Y.S.
      • Kim S.
      • Chae D.W.
      Omacor, n-3 polyunsaturated fatty acid, attenuated albuminuria and renal dysfunction with decrease of SREBP-1 expression and triglyceride amount in the kidney of type II diabetic animals.
      ,
      • Heller J.J.
      • Qiu J.
      • Zhou L.
      Nuclear receptors take center stage in Th17 cell-mediated autoimmunity.
      ,
      • Kaplan M.
      • Aviram M.
      • Hayek T.
      Oxidative stress and macrophage foam cell formation during diabetes mellitus-induced atherogenesis: Role of insulin therapy.
      ,
      • Véret J.
      • Coant N.
      • Berdyshev E.V.
      • Skobeleva A.
      • Therville N.
      • Bailbe D.
      • Gorshkova I.
      • Natarajan V.
      • Portha B.
      • Le Stunff H.
      Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells.
      ,
      • Wang H.
      • Kouri G.
      • Wollheim C.B.
      ER stress and SREBP-1 activation are implicated in beta-cell glucolipotoxicity.
      ,
      • Yano M.
      • Watanabe K.
      • Yamamoto T.
      • Ikeda K.
      • Senokuchi T.
      • Lu M.
      • Kadomatsu T.
      • Tsukano H.
      • Ikawa M.
      • Okabe M.
      • et al.
      Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice.
      ), which translocate to the nucleus and bind to SRE. This leads to induction of iPLA2β transcription and protein expression, which are suppressed in the presence of a dominant negative form of SREBP-1 (
      • Lei X.
      • Bone R.N.
      • Ali T.
      • Zhang S.
      • Bohrer A.
      • Tse H.M.
      • Bidasee K.R.
      • Ramanadham S.
      Evidence of contribution of iPLA2β-mediated events during islet beta-cell apoptosis due to proinflammatory cytokines suggests a role for iPLA2β in T1D development.
      ,
      • Lei X.
      • Zhang S.
      • Barbour S.E.
      • Bohrer A.
      • Ford E.L.
      • Koizumi A.
      • Papa F.R.
      • Ramanadham S.
      Spontaneous development of endoplasmic reticulum stress that can lead to diabetes mellitus is associated with higher calcium-independent phospholipase A2 expression: a role for regulation by SREBP-1.
      ,
      • Lei X.
      • Zhang S.
      • Bohrer A.
      • Barbour S.E.
      • Ramanadham S.
      Role of calcium-independent phospholipase A2beta in human pancreatic islet beta-cell apoptosis.
      ). Intriguingly, the iPLA2β gene exhibits remarkable cross-species homology in the promoter region, which contains putative consensus sequences for a number of stress-related transcriptional factors, suggesting that the iPLA2β gene is a candidate for modulation during periods of stress. Confirmation of iPLA2β induction by these stress-related transcriptional factors will lead to a better understanding of the role of iPLA2β in stress responses and disease manifestation.

      iPLA2β inactivation.

      Inhibitors of iPLA2β include arachidonyl trifluoromethyl ketone (AACOCF3), methyl arachidonyl fluorophosphonate, and palmitoyl trifluoromethyl ketone (PACOCF3), which are sometimes used for “selective” inhibition of cPLA2 (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones.
      ,
      • Magrioti V.
      • Kokotos G.
      Phospholipase A2 inhibitors as potential therapeutic agents for the treatment of inflammatory diseases.
      ,
      • Schaeffer E.L.
      • Gattaz W.F.
      Inhibition of calcium-independent phospholipase A2 activity in rat hippocampus impairs acquisition of short- and long-term memory.
      ). While siRNAs directed at iPLA2β and, now available, iPLA2β-KO and iPLA2β-Tg mice (
      • Bao S.
      • Jacobson D.A.
      • Wohltmann M.
      • Bohrer A.
      • Jin W.
      • Philipson L.H.
      • Turk J.
      Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2β in pancreatic β-cells and in iPLA2β-null mice.
      ,
      • Bao S.
      • Miller D.J.
      • Ma Z.
      • Wohltmann M.
      • Eng G.
      • Ramanadham S.
      • Moley K.
      • Turk J.
      Male mice that do not express group VIA phospholipase A2 produce spermatozoa with impaired motility and have greatly reduced fertility.
      ,
      • Bao S.
      • Song H.
      • Wohltmann M.
      • Ramanadham S.
      • Jin W.
      • Bohrer A.
      • Turk J.
      Insulin secretory responses and phospholipid composition of pancreatic islets from mice that do not express group VIA phospholipase A2 and effects of metabolic stress on glucose homeostasis.
      ) have provided insight into biological processes impacted by iPLA2β, the majority of studies to assess the role of the iPLA2β isoform have utilized a selective inhibitor of iPLA2 (
      • Lei X.
      • Barbour S.E.
      • Ramanadham S.
      Group VIA Ca2+-independent phospholipase A2 (iPLA2β) and its role in beta-cell programmed cell death.
      ). This inhibitor, (E)-6-(bromo-methylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, was synthesized in 1991 and was originally designated as a haloenol lactone substrate (
      • Hazen S.L.
      • Zupan L.A.
      • Weiss R.H.
      • Getman D.P.
      • Gross R.W.
      Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium- dependent and -independent phospholipases A2.
      ), but is now referred to as BEL. The BEL compound is an irreversible inhibitor that selectively targets iPLA2 enzymes and has little or no effect on cPLA2 or sPLA2 activity at concentrations that inhibit iPLA2β or iPLA2γ (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ,
      • Hazen S.L.
      • Zupan L.A.
      • Weiss R.H.
      • Getman D.P.
      • Gross R.W.
      Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium- dependent and -independent phospholipases A2.
      ,
      • Ma Z.
      • Ramanadham S.
      • Hu Z.
      • Turk J.
      Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2.
      ). Over the years, BEL has been used to discern the involvement of iPLA2 in biological processes and, to date, is still considered the only available specific irreversible inhibitor of iPLA2. Because the S- and R-enantiomers of BEL exhibit selective inhibition of iPLA2β and iPLA2γ, respectively (
      • Jenkins C.M.
      • Han X.
      • Mancuso D.J.
      • Gross R.W.
      Identification of calcium-independent phospholipase A2 (iPLA2)beta, and not iPLA2gamma, as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s.
      ), comparison of outcomes using racemic and enantiomers of BEL facilitates distinction of effects due to the β versus the γ isoform. Studies of the mechanism of inhibition reveal that the binding of BEL to iPLA2β leads to generation of a diffusible bromoketomethyl acid, which promotes covalent modification of cysteine residues and not the active site serine of iPLA2β (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones.
      ,
      • Hazen S.L.
      • Zupan L.A.
      • Weiss R.H.
      • Getman D.P.
      • Gross R.W.
      Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium- dependent and -independent phospholipases A2.
      ,
      • Song H.
      • Ramanadham S.
      • Bao S.
      • Hsu F.F.
      • Turk J.
      A bromoenol lactone suicide substrate inactivates group VIA phospholipase A2 by generating a diffusible bromomethyl keto acid that alkylates cysteine thiols.
      ,
      • Song H.
      • Rohrs H.
      • Tan M.
      • Wohltmann M.
      • Ladenson J.H.
      • Turk J.
      Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.
      ). In the presence of DTT, C651 was the only cysteine residue that was modified by BEL, leading to the suggestion that DTT protects iPLA2β from inactivation by BEL. However, using different isolation protocols and higher iPLA2β-specific activity, an additional interaction between C651 and active site serine, S465, was suggested to account for substantial BEL-mediated inhibition (
      • Jenkins C.M.
      • Yang J.
      • Gross R.W.
      Mechanism-based inhibition of iPLA2β demonstrates a highly reactive cysteine residue (C651) that interacts with the active site: mass spectrometric elucidation of the mechanisms underlying inhibition.
      ). While the use of BEL continues to enhance our knowledge of iPLA2β-mediated effects, its irreversible inhibitory profile, potential cytotoxicity (
      • Fuentes L.
      • Perez R.
      • Nieto M.L.
      • Balsinde J.
      • Balboa M.A.
      Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2.
      ,
      • Wilkins 3rd, W.P.
      • Barbour S.E.
      Group VI phospholipases A2: homeostatic phospholipases with significant potential as targets for novel therapeutics.
      ), and several examples of inhibition of nonPLA2 enzymes (
      • van Tienhoven M.
      • Atkins J.
      • Li Y.
      • Glynn P.
      Human neuropathy target esterase catalyzes hydrolysis of membrane lipids.
      ,
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.
      ,
      • Fuentes L.
      • Perez R.
      • Nieto M.L.
      • Balsinde J.
      • Balboa M.A.
      Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2.
      ,
      • Balsinde J.
      • Dennis E.A.
      Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages.
      ,
      • Daniels S.B.
      • Cooney E.
      • Sofia M.J.
      • Chakravarty P.K.
      • Katzenellenbogen J.A.
      Haloenol lactones. Potent enzyme-activated irreversible inhibitors for alpha-chymotrypsin.
      ) render it unfeasible for in vivo iPLA2 inhibition.
      To improve selectivity and reduce toxicity, other compounds are being developed and fluoroketone (FK)-based inhibitors are proving to be as potent as BEL, while being more selective for iPLA2β and also exhibiting reversible inhibitory kinetics (
      • Dennis E.A.
      • Cao J.
      • Hsu Y.H.
      • Magrioti V.
      • Kokotos G.
      Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.
      ,
      • Ali T.
      • Kokotos G.
      • Magrioti V.
      • Bone R.N.
      • Mobley J.A.
      • Hancock W.
      • Ramanadham S.
      Characterization of FKGK18 as inhibitor of group VIA Ca2+-independent phospholipase A2 (iPLA2β): candidate drug for preventing beta-cell apoptosis and diabetes.
      ,
      • Kokotos G.
      • Hsu Y.H.
      • Burke J.E.
      • Baskakis C.
      • Kokotos C.G.
      • Magrioti V.
      • Dennis E.A.
      Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.
      ). Because FK inhibitors target serine active sites, they could potentially also inhibit cPLA2s. However, modification of the FK group along with addition of a hydrophobic terminus, connected by a medium-length carbon chain to mimic the fatty acid chain, conferred selectivity of the FK compounds for iPLA2 versus sPLA2 or cPLA2 (
      • Kokotos G.
      • Hsu Y.H.
      • Burke J.E.
      • Baskakis C.
      • Kokotos C.G.
      • Magrioti V.
      • Dennis E.A.
      Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.
      ), and earlier generation FK compounds (FKGK11 and FKGK2) were found to be beneficial in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis (
      • Kalyvas A.
      • Baskakis C.
      • Magrioti V.
      • Constantinou-Kokotou V.
      • Stephens D.
      • Lopez-Vales R.
      • Lu J.Q.
      • Yong V.W.
      • Dennis E.A.
      • Kokotos G.
      • et al.
      Differing roles for members of the phospholipase A2 superfamily in experimental autoimmune encephalomyelitis.
      ). Subsequently, the FK-based inhibitor of iPLA2β (FKGK18) was found to be 7-fold more potent than FKGK11 toward iPLA2β 195 and >455 times more potent for iPLA2β than for group IVA cPLA2 and group V sPLA2, respectively (
      • Stephens D.
      • Barbayianni E.
      • Constantinou-Kokotou V.
      • Peristeraki A.
      • Six D.A.
      • Cooper J.
      • Harkewicz R.
      • Deems R.A.
      • Dennis E.A.
      • Kokotos G.
      Differential inhibition of group IVA and group VIA phospholipases A2 by 2-oxoamides.
      ); and effective in cell-based studies (
      • Gil-de-Gómez L.
      • Astudillo A.M.
      • Guijas C.
      • Magrioti V.
      • Kokotos G.
      • Balboa M.A.
      • Balsinde J.
      Cytosolic group IVA and calcium-independent group VIA phospholipase A2s act on distinct phospholipid pools in zymosan-stimulated mouse peritoneal macrophages.
      ) and in countering T1D (
      • Bone R.N.
      • Gai Y.
      • Magrioti V.
      • Kokotou M.G.
      • Ali T.
      • Lei X.
      • Tse H.M.
      • Kokotos G.
      • Ramanadham S.
      Inhibition of Ca2+-independent phospholipase A2beta (iPLA2β) ameliorates islet infiltration and incidence of diabetes in NOD mice.
      ). Recently developed and awaiting characterization is an even more selective inhibitor (GK187) of iPLA2β (
      • Magrioti V.
      • Nikolaou A.
      • Smyrniotou A.
      • Shah I.
      • Constantinou-Kokotou V.
      • Dennis E.A.
      • Kokotos G.
      New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2.
      ). On-going deuterium exchange mass spectrometry and molecular dynamics analyses suggest that FKGK inhibitor binding to iPLA2β causes changes in the loops surrounding the active site of iPLA2β in the catalytic domain, blocking access to phospholipid substrates and reducing solvent accessibility (
      • Hsu Y.H.
      • Bucher D.
      • Cao J.
      • Li S.
      • Yang S.W.
      • Kokotos G.
      • Woods Jr, V.L.
      • McCammon J.A.
      • Dennis E.A.
      Fluoroketone inhibition of Ca2+-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.
      ). As the development of chemical inhibitors continues, newer structurally dissimilar and smaller compounds (
      • Barbayianni E.
      • Stephens D.
      • Grkovich A.
      • Magrioti V.
      • Hsu Y.H.
      • Dolatzas P.
      • Kalogiannidis D.
      • Dennis E.A.
      • Kokotos G.
      2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides.
      ) with even greater selectivity for iPLA2β are forthcoming, as described at the 6th International Conference on PLA2s in 2015 (Kokotos et al., unpublished observations).

      Proposed roles for iPLA2β

      Membrane remodeling.

      One of the earliest proposed functions for iPLA2β was a “housekeeping” role that involves generation of lysophospholipid acceptors for incorporation of AA into phospholipids, based on experiments involving inhibition of iPLA2β activity in P388D1 cells with BEL or with an antisense oligonucleotide (
      • Balsinde J.
      • Balboa M.A.
      • Dennis E.A.
      Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages.
      ,
      • Balsinde J.
      • Bianco I.D.
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.
      ). Inhibition of iPLA2β activity in P388D1 cells suppressed (∼60%) incorporation of [3H]AA into phospholipids while reducing (∼60%) [3H]LPC levels in [3H]choline-labeled P388D1 cells. However, [3H]palmitic acid incorporation was only slightly reduced. This is thought to represent the mechanism whereby iPLA2β inhibition reduces incorporation of [3H]AA into P388D1 cell phospholipids. Such incorporation reflects a deacylation/reacylation cycle (
      • Chilton F.H.
      • Fonteh A.N.
      • Surette M.E.
      • Triggiani M.
      • Winkler J.D.
      Control of arachidonate levels within inflammatory cells.
      ) of phospholipid remodeling rather than de novo synthesis (
      • Dennis E.A.
      The biosynthesis of phospholipids.
      ), and the level of LPC acceptors is thought to limit the rate of [3H]AA incorporation into P388D1 cell PC (
      • Balsinde J.
      • Balboa M.A.
      • Dennis E.A.
      Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages.
      ,
      • Balsinde J.
      • Bianco I.D.
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.
      ).
      A second housekeeping function for iPLA2β is suggested from studies with CTP:PC cytidyltransferase (CT)-overexpressing Chinese hamster ovary cells (
      • Barbour S.E.
      • Kapur A.
      • Deal C.L.
      Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.
      ). CT catalyzes the rate-limiting step in PC biosynthesis via the Kennedy pathway, and cells overexpressing CT exhibit increased rates of PC biosynthesis and degradation and little net change in PC accumulation (
      • Barbour S.E.
      • Kapur A.
      • Deal C.L.
      Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.
      ). Immunoreactive iPLA2β protein and activity increase in the CT overexpressors and the increased PC degradation is prevented by BEL, suggesting that iPLA2β is upregulated in response to CT overexpression (
      • Barbour S.E.
      • Kapur A.
      • Deal C.L.
      Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.
      ). In general, this could represent an important role for iPLA2β in cell biology because PC biosynthesis is involved in regulation of cell cycle and apoptosis (
      • Wolf B.A.
      • Pasquale S.M.
      • Turk J.
      Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion.
      ).
      More recently, in a study examining the effects of lipotoxicity in β-cells, the monolysocardiolipin content was reported to correlate with iPLA2β expression level (
      • Song H.
      • Wohltmann M.
      • Tan M.
      • Ladenson J.H.
      • Turk J.
      Group VIA phospholipase A2 mitigates palmitate-induced beta-cell mitochondrial injury and apoptosis.
      ). The authors suggested that iPLA2β contributed to cardiolipin remodeling by excising oxidized PUFA residues from cardiolipin to yield monolysocardiolipin species for reacylation with unoxidized C18:2-CoA to regenerate the native cardiolipin structure and function. This facilitated stabilization of association of cytochrome c with mitochondrial membranes and decreasing its appearance in the cytosol, thereby reducing ROS-mediated apoptosis. The authors concluded that participation of iPLA2β in such an excision-reacylation mechanism of repair of oxidized phospholipids represents a special case of the originally proposed function of the enzyme in phospholipid remodeling.

      Cell proliferation.

      Studies utilizing chemical inhibition or genetic modification protocols reveal a positive correlation between maintenance of iPLA2β activity and cell proliferation. In the presence of BEL, human promonocytic U937 (
      • Balboa M.A.
      • Sáez Y.
      • Balsinde J.
      Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes.
      ) and ovarian carcinoma (
      • Song Y.
      • Wilkins P.
      • Hu W.
      • Murthy K.S.
      • Chen J.
      • Lee Z.
      • Oyesanya R.
      • Wu J.
      • Barbour S.E.
      • Fang X.
      Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.
      ) cells exhibit a decreased rate of proliferation and this is rescued in Caco-2 (
      • Sanchez T.
      • Moreno J.J.
      Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth.
      ) and endothelial (
      • Herbert S.P.
      • Walker J.H.
      Group VIA calcium-independent phospholipase A2 mediates endothelial cell S phase progression.