Possible antithrombotic effects of Angelica keiskei (Ashitaba).

Angelica keiskei Koidzumi (Ashitaba) is a large perennial herb that is native to the Pacific coast of Japan, and it has recently become popular as herbal medicine, dietary supplement and health food in Asian countries. The structures of various constituents isolated from Ashitaba such as chalcones, flavanones and coumarins have been precisely characterized, and many of them have bioactivities. A recent study clarified that Angelica keiskei exerts actions that lead to the prevention of thrombosis. Here, we introduce the possibility that ingesting Ashitaba could help to prevent thrombotic diseases.

Anti-platelet effects of chalcones from Angelica keiskei Koidzumi (Ashitaba) in vivo.

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine that is also regarded in Japan as a health food with potential antithrombotic properties. The ability of the major chalcones, xanthoangelol (XA) and 4-hydroxyderricin (4-HD) extracted from Ashitaba roots to inhibit platelet aggregation activity in vitro was recently determined. However, the anti-platelet activities of Ashitaba chalcones in vivo have remained unclear. The present study examines the anti-platelet effects of Ashitaba exudate and its constituent chalcones using mouse tail-bleeding models that reflect platelet aggregation in vivo. Ashitaba exudate and the major chalcone subtype XA, suppressed the lipopolysaccharide (LPS)-induced shortening of mouse tail bleeding. However, trace amounts of other Ashitaba chalcone subtypes including xanthoangelols B (XB), D (XD), E (XE) and F (XF) did not affect tail bleeding. These results suggest that the major chalcone subtype in Ashitaba, XA, has anti-platelet-activities in vivo.

Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean.

Helper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

CLOCK is involved in obesity-induced disordered fibrinolysis in ob/ob mice by regulating PAI-1 gene expression.

BACKGROUND: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. AIM: The present study investigates whether the circadian clock component CLOCK is involved in obesity-induced PAI-1 elevation. METHODS: We examined plasma PAI-1 and mRNA expression levels in tissues from leptin-deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. RESULTS: Our results demonstrated that plasma PAI-1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI-1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI-1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI-1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. CONCLUSION: These observations suggest that CLOCK is involved in obesity-induced disordered fibrinolysis by regulating PAI-1 gene expression in a tissue-dependent manner. Furthermore, it appears that obesity-induced PAI-1 production in adipose tissue is not closely related to systemic PAI-1 increases in vivo.

Enhancement of mast cell survival: a novel function of some secretory phospholipase A(2) isotypes.

This study tested the hypothesis that certain secretory phospholipase A(2) (sPLA(2)) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA(2) activity and sPLA(2) receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA(2), prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA(2) hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA(2) did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA(2). Additionally, both wild-type and catalytically inactive group IB PLA(2) induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin V(FITC) binding and only partially inhibited thymidine incorporation in sPLA(2)-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin V(FITC) binding and thymidine incorporation in mast cells maintained with IL-3. sPLA(2) enhanced phosphoinositide 3'-kinase activity, and a specific inhibitor of phosphoinositide 3'-kinase reversed the antiapoptotic effects of sPLA(2). Likewise, sPLA(2) increased the degradation of I-kappaBalpha, and specific inhibitors of nuclear factor kappa activation (NF-kappaB) reversed the antiapoptotic effects of sPLA(2). Together, these experiments reveal that certain isotypes of sPLA(2) enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA(2) receptors rather than sPLA(2) catalytic products.

Early de novo gene expression is required for 15-deoxy-Delta 12,14-prostaglandin J2-induced apoptosis in breast cancer cells.

Cyclopentenone prostaglandin derivatives of arachidonic acid are potent inducers of apoptosis in a variety of cancer cell types. Several investigators have shown that the terminal derivative of prostaglandin J(2) (PGJ(2)) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), but 15dPGJ(2) effects can be mediated by PPARgamma-dependent and PPARgamma-independent mechanisms. Here we report that 15dPGJ(2) regulates early gene expression critical to apoptosis. Specifically, 15dPGJ(2) induces potent and irreversible S phase arrest that is correlated with expression of genes critical to cell cycle arrest and apoptosis, including the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Inhibition of RNA or protein synthesis abrogates apoptosis induced by 15dPGJ(2) in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor-alpha or CD95/Fas ligand. Additionally, 15dPGJ(2) induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ(2)-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

Magnitude of peroxisome proliferator-activated receptor-gamma activation is associated with important and seemingly opposite biological responses in breast cancer cells.

BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARgamma leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARgamma agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors. METHODS: We studied the effects of low-, moderate-, and high-dose treatment of the PPARgamma ligands 15-deoxy-delta1214 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231. RESULTS: The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARgamma activation. For example, low concentrations of 15dPGJ2 (<2.5 microM) and TGZ (<5 microM) increased cellular proliferation, but concentrations of 15dPGJ2 > or = 10 microM and of TGZ at 100 microM blocked cell growth. TGZ (100 microM) slowed cell cycle progression, and 15dPGJ2 (10 microM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 microM 15dPGJ2 or 5 to 100 microM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARgamma ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis. CONCLUSIONS: PPARgamma activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARgamma activation. These data may explain, at least in part, some of the discordant results previously reported.

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells.

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.

Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts.

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.

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.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp(522) during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA(2)(1-522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A(2)s (PLA(2)s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA(2)s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA(2)(1-522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca(2+)-independent PLA(2) (iPLA(2); type VI), a phospholipid remodeling PLA(2), contributes to the cell death-associated increase in fatty acid release. iPLA(2) was cleaved at Asp(183) by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA(2) functions. iPLA(2) had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA(2)alpha behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA(2) accelerates phospholipid turnover that may lead to apoptotic membranous changes.

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Characterization of heparin low-affinity phospholipase A1 present in brain and testicular tissue.

We identified a unique phospholipase A (PLA) with relatively low heparin affinity, which was distinguishable from the heparin-binding secretory PLA2s, in rat, mouse, and bovine brains and testes. The partially purified enzyme was Ca2+-independent at neutral pH but Ca2+-dependent at alkaline pH. It predominantly hydrolyzed phosphatidic acid (PA) in the presence of Triton X-100 and phosphatidylethanolamine (PE) in its absence. When rat brain-derived endogenous phospholipids were used as a substrate, the enzyme released saturated fatty acids in marked preference to unsaturated ones. Consistent with this observation, the enzyme hydrolyzed sn-1 ester bonds in the substrates about 2,000 times more efficiently than sn-2 ones, thereby acting like PLA1. The enzyme also exhibited weak but significant sn-1 lysophospholipase activity. On the basis of its limited tissue distribution, substrate head group specificity and immunochemical properties, this enzyme appears to be identical to the recently cloned PA-preferring PLA1.

Influence of coenzyme A-independent transacylase and cyclooxygenase inhibitors on the proliferation of breast cancer cells.

Recent studies have demonstrated that arachidonic acid (AA) may serve as an important signal that blocks cell proliferation of certain neoplastic cells. The current study was conducted to determine whether disruption of AA homeostasis influences breast cancer cell proliferation and death. Initial experiments revealed that inhibition of AA remodeling through membrane phospholipids by inhibitors of the enzyme, coenzyme A-independent transacylase (CoA-IT), attenuates the proliferation of the estrogen receptor-negative, MDA-MB-231, and estrogen receptor-positive, MCF-7 breast cancer cell lines. This growth inhibition was accompanied by a marked accumulation of AA in both free fatty acid and triglyceride forms, a marker of intracellular AA stress within mammalian cells. Cell cycle synchronization experiments revealed that the CoA-IT inhibitor, SB-98625, blocked MDA-MB-231 cell replication in early to mid G1 phase. Time-lapse video microscopy, used to observe the changes in cell morphology associated with apoptosis, indicated that SB-98625 treatment induced early rounding and occasional blebbing but not late apoptotic events, blistering, and lysis. The cyclooxygenase inhibitors, NS-398 and indomethacin, were found to be less potent blockers of cell proliferation and poor inducers of cellular AA accumulation than CoA-IT inhibitors in these breast cancer cell lines. Finally, AA provided exogenously blocked the proliferation of MCF-7 cells, and this effect could be attenuated in MCF-7 cells overexpressing the glutathione peroxidase gene, GSHPx-1. Taken together, these experiments suggest that disruption of AA remodeling in a manner that increases intracellular AA may represent a novel therapeutic strategy to reduce cancer cell proliferation and that an oxidized AA metabolite is likely to mediate this effect.

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 downward arrowX), 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.

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)-1beta and tumor necrosis factor alpha (TNFalpha) 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-1beta/TNFalpha-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 PLA2 enzymes 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 cPLA2 and COX-2 expression via endogenous PGE2.

Regulatory functions of phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGa) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into there large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca(2+)-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general understanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

The perturbed membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A2 to liberate arachidonic acid.

Several lines of evidence have suggested that the plasma membranes of cells elicited by proinflammatory stimuli or microvesicles shed from activated cells are sensitive to extracellular type II secretory phospholipase A2 (sPLA2) that liberates fatty acids and lysophospholipids. Here we report that the membranes of cells undergoing apoptosis are highly susceptible to type II sPLA2. When neuronally differentiated rat pheochromocytoma PC12 cells deprived of nerve growth factor and serum, mouse mast cells deprived of hematopoietic cytokines or human monocytic U937 cells stimulated via Fas antigen (a receptor for the death factor Fas ligand), were exposed to type II sPLA2 at concentrations comparable to those detected at inflamed sites, the release of arachidonic acid was significantly accelerated in association with the process of programmed cell death. Arachidonic acid release by sPLA2 was dependent on the extracellular Ca2+ and was accompanied by preferential hydrolysis of phosphatidylethanolamine and phosphatidylserine in the membrane phospholipids. Association of sPLA2 with cell surface proteoglycan, which has been shown to be a prerequisite for endogenous sPLA2-dependent arachidonic acid release from the plasma membranes of live cells, was not essential for sPLA2-mediated hydrolysis of apoptotic cell membranes. Taking these results together, the apoptotic cell membrane is a potential target for extracellular type II sPLA2. The present findings may be relevant to events occurring at inflammatory or ischemic disease sites where apoptotic cells accumulate.

Type II phospholipase A2 is linked to cyclooxygenase-2-mediated delayed prostaglandin D2 generation by cultured mouse mast cells following FcepsilonRI- and cytokine-dependent activation.

Treatment of murine bone marrow-derived mast cells (BMMC) with interleukin (IL)-10 and IL-1beta increased the expression of type II secretory phospholipase A2 (sPLA2), but not cytosolic PLA2 (cPLA2), after culture for several hours. Further stimulation with IgE and antigen (IgE/Ag) increased cyclooxygenase (COX)-2 expression dramatically, accompanied by augmented delayed prostaglandin (PG) D2 generation over several hours. BMMC were also found to express type IIC PLA2 (PLA2-IIC), a recently described novel sPLA2 possessing 16 cysteine residues, expression of which changed minimally after BMMC activation. Delayed PGD2 generation was suppressed by approximately 80% by an antibody raised against recombinant murine type II sPLA2. These results suggest that, of the three PLA2s expressed in BMMC, type II sPLA2 is the critical enzyme that is coupled with COX-2-dependent PGD2 generation elicited by IgE/Ag in the presence of IL-10 + IL-1beta.