Hiroshi Kuwata

Cytosolic Phospholipase A2 Is Required for Cytokine-induced Expression of Type IIA Secretory Phospholipase A2 That Mediates Optimal Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Rat 3Y1 Fibroblasts

Activation of rat fibroblastic 3Y1 cells with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induced delayed prostaglandin (PG) E2 generation over 6–48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2(cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1β/TNFα-induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2generation. Some lipoxygenase-derived products might be involved in this event, since IL-1β/TNFα-induced type IIA sPLA2induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1β/TNFα treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes.

Functional Association of Type IIA Secretory Phospholipase A2 with the Glycosylphosphatidylinositol-anchored Heparan Sulfate Proteoglycan in the Cyclooxygenase-2-mediated Delayed Prostanoid-biosynthetic Pathway

An emerging body of evidence suggests that type IIA secretory phospholipase A2(sPLA2-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA2-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA2-IIA. sPLA2-IIA-dependent PGE2 generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA2-IIA. Overexpression of glypican-1 increased the association of sPLA2-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA2-IIA. Glypican-1 overexpression led to marked augmentation of sPLA2-IIA-mediated arachidonic acid release, PGE2 generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA2-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA2-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA2-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.

Prostaglandin E2 Amplifies Cytosolic Phospholipase A2- and Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Mouse Osteoblastic Cells ENHANCEMENT BY SECRETORY PHOSPHOLIPASE A2

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1β and tumor necrosis factor α (TNFα) was dependent upon cytosolic phospholipase A2(cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1β/TNFα-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2and COX-2 expression via endogenous PGE2.

Cellular components that functionally interact with signaling phospholipase A(2)s.

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.

Studies on a Mechanism by Which Cytosolic Phospholipase A2 Regulates the Expression and Function of Type IIA Secretory Phospholipase A2

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.

Group VIB Ca2+-independent Phospholipase A2γ Promotes Cellular Membrane Hydrolysis and Prostaglandin Production in a Manner Distinct from Other Intracellular Phospholipases A2

Although group VIA Ca2+-independent phospholipase A2β (iPLA2β) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2γ. Lentiviral transfection of iPLA2γ into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2γ small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2γ. iPLA2γ protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2γ and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2γ into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2γ in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2β and iPLA2γ in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2γ to tumorigenesis.

A Novel Role of Group VIB Calcium-independent Phospholipase A2 (iPLA2γ) in the Inducible Expression of Group IIA Secretory PLA2 in Rat Fibroblastic Cells

Group IIA secretory phospholipase A2 (sPLA2-IIA) is a prototypic sPLA2 enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA2 by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA2 (cPLA2α) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA2α is required for de novo induction of sPLA2. However, because of the broad specificity of several cPLA2α inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA2-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA2 (iPLA2γ), rather than cPLA2α, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA2-IIA in cPLA2α knockdown cells, in which cPLA2α protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA2γ, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA2, markedly reduced the cytokine-induced expression of sPLA2-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA2γ inhibitor, suppressed the cytokine-induced sPLA2-IIA expression, whereas (S)-bromoenol lactone, an iPLA2β inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA2-IIA expression was also abolished by knockdown of iPLA2γ. These findings open new insight into a novel regulatory role of iPLA2γ in stimulus-coupled sPLA2-IIA expression.

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

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

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

Group VIB Calcium-Independent Phospholipase A2 (iPLA2γ) Regulates Platelet Activation, Hemostasis and Thrombosis in Mice

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