Yoshihito Nakatani

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE2 biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr9), which is known to be critical for the activity of cytosolic GSHS-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE2 that plays a role in maintenance of tissue homeostasis.

Prostaglandin E synthase

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin (PG)H2 to PGE2, occurs in multiple forms with distinct enzymatic properties, modes of expression, cellular and subcellular localizations and intracellular functions. Cytosolic PGES (cPGES) is a cytosolic protein that is constitutively expressed in a wide variety of cells and tissues and is associated with heat shock protein 90 (Hsp90). Membrane-associated PGES (mPGES), the expression of which is stimulus-inducible and is downregulated by anti-inflammatory glucocorticoids, is a perinuclear protein belonging to the microsomal glutathione S-transferase (GST) family. These two PGESs display distinct functional coupling with upstream COXs in cells; cPGES is predominantly coupled with the constitutive COX-1, whereas mPGES is preferentially linked with the inducible COX-2. Several cytosolic GSTs also have the capacity to convert PGH2 to PGE2 in vitro. Accumulating evidence has suggested that mPGES participates in various pathophysiological states in which COX-2 is involved, implying that mPGES represents a potential novel target for drug development.

Fas-induced Arachidonic Acid Release Is Mediated by Ca2+-independent Phospholipase A2 but Not Cytosolic Phospholipase A2, Which Undergoes Proteolytic Inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD↓X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.

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.

Type II Secretory Phospholipase A2 Associated with Cell Surfaces via C-terminal Heparin-binding Lysine Residues Augments Stimulus-initiated Delayed Prostaglandin Generation

Type II secretory phospholipase A2 (sPLA2) has been shown to be induced by a variety of proinflammatory stimuli and, therefore, has been implicated in the inflammatory process. In order to determine whether association of sPLA2 with cell surfaces via heparan sulfate proteoglycan is important for its effects on cellular functions, we have identified the critical domain in sPLA2 for heparin and cell surface binding and examined its role in cellular prostaglandin (PG) biosynthesis. Replacement of several conserved Lys residues in the C-terminal region of mouse and rat sPLA2s by Glu resulted in a marked reduction of their capacities to bind to heparin and mammalian cell surfaces without affecting their enzymatic activities toward dispersed phospholipid as a substrate. CHO cells stably transfected with wild-type sPLA2 released about twice as much arachidonic acid (AA) during culture for 10 h with fetal calf serum and interleukin-1β than cells transfected with vector alone, whereas the ability to enhance AA release was impaired in sPLA2 mutants incapable of binding to cell surfaces. AA released by wild-type sPLA2-transfected CHO cells was metabolized to prostaglandin E2 via prostaglandin endoperoxide H synthase (PGHS)-2 after IL-1β stimulation, revealing a particular functional linkage of sPLA2 to PGHS-2. In contrast, A23187-initiated immediate AA release over 30 min was not affected by sPLA2 overexpression. Taken together, these results suggest that sPLA2 expressed endogenously and anchored on cell surfaces via its C-terminal heparin-binding domain is involved in the PGHS-2-dependent delayed PG biosynthesis initiated by growth factors and cytokines during long term culture.

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.

Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models.

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H(2) (PGH(2)) to PGE(2), is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE(2) production. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.

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.

Identification of a Cellular Protein That Functionally Interacts with the C2 Domain of Cytosolic Phospholipase A2α

Cytosolic phospholipase A2(cPLA2) α plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA2α in a Ca2+-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA2α and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA2α and vimentin colocalized around the perinuclear area in cPLA2α-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA2α to function properly during the eicosanoid-biosynthetic process.

Phospholipase A2 mediates ischemic injury in the hippocampus: a regional difference of neuronal vulnerability.

Although it is well known that the hippocampal CA1 subfield is highly vulnerable to ischemic injury, cellular mechanisms leading to this neuronal degeneration are not fully understood. Using organotypic cultures of rat hippocampal slices, we determined whether phospholipase A2 (PLA2) is activated in response to ischemic conditions (OGD; oxygen and glucose deprivation). The PLA2 activity in the pyramidal cell layer increased immediately following a 35‐min exposure to OGD, which was likely to be mediated by selective activation of cytosolic Ca2+‐dependent PLA2 subtype (cPLA2). This enhancement lasted for at least 24 h. Interestingly, no apparent increase was detected in the dentate gyrus. Twenty‐four hours after the OGD exposure, neuronal death was detected mainly in the CA1 region of hippocampal slices. To examine whether the PLA2 activation is causally or protectively involved in the ischemic injury, we investigated the effect of pharmacological blockade of PLA2 on the OGD‐induced neuronal death. The PLA2 inhibitor bromophenacyl bromide efficiently prevented the cell death in a concentration‐dependent manner. Similar results were obtained for the selective cPLA2 inhibitor AACOCF3. However, the Ca2+‐independent PLA2 inhibitor bromoenol lactone and the secretory PLA2 inhibitor LY311727 were virtually ineffective. These results suggest that cPLA2 plays a causative role in the neuronal death following OGD exposure. Thus, the present study may provide novel therapeutic targets for the development of neuroprotective agents.