Sequential treatment with zoledronic acid followed by teriparatide or vice versa increases bone mineral density and bone strength in ovariectomized rats.

Bisphosphonates (BPs) and teriparatide (TPTD) are both effective treatments for osteoporosis, but BP treatment prior to daily TPTD treatment has been shown to impair the effect of TPTD in some clinical studies. In contrast, the loss of bone mineral density (BMD) that occurs after withdrawal of TPTD can be prevented by BP treatment. Although various studies have investigated the combination and/or sequential use of BP and TPTD, there have been no clinical studies investigating sequential treatment with zoledronic acid (ZOL) and TPTD (or vice versa). In this study, we evaluated the effects of sequential treatment with TPTD followed by ZOL, and ZOL followed by TPTD, using ovariectomized (OVX) rats. Two months after OVX, osteopenic rats were treated with ZOL, TPTD, or vehicle for a period of 4 months (first treatment period), and then the treatments were switched and administered for another 4 months (second treatment period). The group treated with ZOL followed by TPTD showed an immediate increase in BMD of the proximal tibia and greater BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur than the group treated with ZOL followed by vehicle. Serum osteocalcin, a marker of bone formation, increased rapidly after switching to TPTD from ZOL. The group treated with TPTD followed by ZOL did not lose BMD in the proximal tibia after TPTD was stopped, while the group treated with TPTD followed by vehicle did lose BMD. The BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur were greater in the group treated with TPTD followed by ZOL than in the group treated with TPTD followed by vehicle. The increase in serum osteocalcin and urinary CTX after withdrawal of TPTD was prevented by the switch from TPTD to ZOL. In conclusion, our results demonstrate that switching from ZOL to TPTD resulted in a non-attenuated anabolic response in the lumbar spine and femur of OVX rats. In addition, switching from TPTD to ZOL caused BMD to be maintained or further increased. If these results can be reproduced in a clinical setting, the sequential use of ZOL followed by TPTD or vice versa in the treatment of osteoporosis patients would contribute to increases in BMD that, hopefully, would translate into a corresponding decrease in the incidence of vertebral and non-vertebral fractures.

Expression of type V secretory phospholipase A in myocardial remodelling after infarction.

AIMS: Secretory phospholipase A2 is associated with ischaemic injury in the human heart, but the distribution of type V secretory phospholipase A2 (sPLA2-V) remains unknown. The significance of sPLA2-V in myocardial infarction was investigated histopathologically. METHODS: Sequential changes in the localization of sPLA2-V and its mRNA in myocardial tissues obtained from 30 autopsied hearts were examined by immunohistochemistry and in situ hybridization and compared with those of fibronectin, vascular endothelial growth factor (VEGF), interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and cyclooxygenase-2 (COX-2). RESULTS: No expression of sPLA2-V was observed in normal heart, but it was promptly expressed in wavy myofibres positive for fibronectin just after the onset of infarction. sPLA2-V was subsequently expressed in ischaemic cardiomyocytes around the lesion. The expression decreased at the granulation tissue and disappeared at the chronic stage with scar formation. The distribution of the signal for sPLA2-V mRNA paralleled that of the protein. Ischaemic myocytes around the lesion expressed VEGF, IL-1beta, TNF-alpha and COX-2 at all stages. CONCLUSIONS: sPLA2-V production in myocardium is limited to the acute phase of infarction. sPLA2-V may play a dual role, acting both to remove degraded cell-membrane through cooperative activity with COX-2 in necrotic areas and to attack ischaemic myocytes around the lesion via degradation of membrane phospholipids.

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.

Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.

We have recently shown that two distinct prostaglandin (PG) E(2) synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE(2) biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE(2) synthase, the perinuclear enzymes thromboxane synthase and PGI(2) synthase generated their respective products via COX-2 in preference to COX-1 in both the -induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD(2) synthase preferentially used COX-1 and COX-2 in the -induced immediate and interleukin-1-induced delayed PGD(2)-biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A(2). Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA(2)s at a moment when PG production takes place.

Progress and problems in the prescribing/dispensing split and "divided package sales" by wholesalers.

Given the rapid progress of the prescribing/dispensing split, the ability of pharmacies to obtain and stock a small quantity of pharmaceuticals is seen as essential. Toho Yakuhin's experience in "divided package sales" (wholesalers open an original package supplied by the manufacturer and sell only a part of the contents) shows that the number of orders and sales amount, and the number of pharmacies ordering in sales have remarkably increased. One or two kinds of pharmaceuticals were ordered in 73% cases, and one or two units in about 50%. This implies that "divided package sales" are utilized to obtain rarely prescribed drugs. On the other hand, five or more kinds of pharmaceuticals were ordered in 10% of cases, and ten or more units in 17%. "Divided package sales" were more used for low-price drugs. These indicated that "divided package sales" seemed to be looked upon by pharmacies as a means of obtaining a small quantity of pharmaceuticals, and seemed to be used not only in an emergency but also routinely. There are several problems for wholesalers in operating "divided package sales", such as frequent delivery, delivery cost and information supply. Pharmacies should obtain a small quantity of pharmaceuticals by the routine delivery and should share the delivery cost. Pharmaceutical manufacturers' cooperation in printing necessary information on each immediate container will be useful. Though there could be alternative ways of obtaining a small quantity of pharmaceuticals, all of them have the matter of delivery. We believe "divided package sales" will contribute to this situation.

Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms.

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.

Spectrophotometric determination of saponin in Yucca extract used as food additive.

A spectrophotometric method was developed for the determination of saponin in Yucca extract or its preparation for food additive use. A saponin fraction of Yucca extract was prepared by column chromatography with porous polymer, and hydrolyzed with a 2 mol/L mixture of hydrochloric acid-ethanol (1 + 1) to generate sapogenin. Sapogenin amounts were determined by measuring absorbance at 430 nm, based on the color reactions with anisaldehyde, sulfuric acid, and ethyl acetate. Recoveries from Yucca extracts were 91.5-95.1%, and the detection limit was 10 microg/kg. Commercial Yucca extracts for food additive use were composed of 5.6-6.4% (w/w) saponin, making it a minor component.

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.

Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells.

Cytosolic phospholipase A(2) (cPLA(2)) plays a critical role in mast-cell-related allergic responses [Uozumi, Kume, Nagase, Nakatani, Ishii, Tashiro, Komagata, Maki, Ikuta, Ouchi et al. (1997) Nature (London) 390, 618-622]. Bone-marrow-derived mast cells from mice lacking cPLA(2) (cPLA(-/-)(2) mice) were used in order to better define the role of cPLA(2) in the maturation and degranulation of such cells. Cross-linking of high-affinity receptors for IgE (FcepsilonRI) on cells from cPLA(-/-)(2) mice led to the release of negligible amounts of arachidonic acid or its metabolites, the cysteinyl leukotrienes and prostaglandin D(2), indicating an essential role for cPLA(2) in the production of these allergic and pro-inflammatory lipid mediators. In addition, the histamine content of the mast cells and its release from the cells were reduced to 60%. While these results are in agreement with a reduced anaphylactic phenotype of cPLA(-/-)(2) mice, the ratios of release of histamine and beta-hexosaminidase were, paradoxically, significantly higher for cells from cPLA(-/-)(2) mice than for those from wild-type mice. Consistently, IgE-induced calcium influx in mast cells was greater and more prolonged in cells from cPLA(-/-)(2) mice than in those from wild-type mice. Thus the loss of cPLA(2) not only diminishes the release of lipid mediators, but also alters degranulation. While the overall effect is still a decrease in the release of mast cell mediators, explaining the in vivo findings, the present study proposes a novel link between cPLA(2) and the degranulation machinery.

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells.

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.

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.

Internalization and degradation of type IIA phospholipase A(2) in mast cells.

Whereas exogenous types IB and X secretory phospholipase A(2) (sPLA(2)) elicited prostaglandin D(2) (PGD(2)) production in mouse bone marrow-derived mast cells (BMMC), sPLA(2)-IIA was unable to do so. In search of a mechanism underlying this cellular refractoriness to exogenous sPLA(2)-IIA, we now report that this isozyme is promptly associated with cell surfaces, internalized, and then degraded in BMMC. Adsorption of sPLA(2)-IIA to BMMC was prevented by addition of heparin to the medium. Moreover, a heparin-nonbinding sPLA(2)-IIA mutant did not bind to BMMC. These results indicate that this sPLA(2)-IIA inactivation process depends on its rapid binding to heparan sulfate proteoglycan (HSPG) on BMMC surfaces. Thus, the present observations represent a particular situation in which cell surface HSPG exhibit a negative regulatory effect on cellular function of sPLA(2)-IIA, and argue that HSPG does not always act as a functional adapter for heparin-binding sPLA(2)s in mammalian cells as has been demonstrated before.

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

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) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) 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 (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-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 PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2.

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE(2) biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE(2) generation. Arg(110), a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A(2) allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE(2) biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways.

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.

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.

Augmented prostaglandin E2 generation resulting from increased activities of cytosolic and secretory phospholipase A2 and induction of cyclooxygenase-2 in interleukin-1 beta-stimulated rat calvarial cells during the mineralizing phase.

OBJECTIVE AND DESIGN: To assess prostaglandin (PG) E2 production by osteoblasts during the mineralizing phase after interleukin (IL)-1beta stimulation, using an in vitro system of rat calvarial cells cultured for 21 days. METHODS: The cells, which reached confluence after 3 days, were designated day 0 cells. Culture was continued for a further 21 days after confluence. The cells on the 21st day of the culture were designated day 21 cells. RESULTS: The PGE2 concentration in the medium of the day 21 cells was increased 72 h after IL-1beta treatment, and reached a peak level approximately 1,400 times that of the day 0 cells 6 h after IL-1beta treatment. We examined the effects of IL-1beta on PGE2 production and changes in the relevant enzyme activities, and found that the activities of cytosolic phospholipase A2 (cPLA2), type II secretory PLA2 (sPLA2) and cyclooxygenase (COX)-2 in the day 21 cells were increased. Both selective COX-2 inhibitor and cPLA2 inhibitor abolished PGE2 generation, whereas an sPLA2 inhibitor partially inhibited it. Taken together, these results indicate that COX-2 and cPLA2 play pivotal roles and sPLA2 is involved in IL-1beta-stimulated PGE2 production by these cells. Furthermore, we found that IL-Ibeta treatment induced PGE synthase activity and this correlated well with PGE2 production. CONCLUSION: Augmented PGE2 production by mineralizing osteoblasts after IL-1beta treatment, and the involvement of IL-1beta-induced cPLA2, sPLA2, COX-2 and PGE synthase activities in this phenomenon were demonstrated.

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.