Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha) is the only PLA(2) that exhibits specificity for sn-2 arachidonic acid consistent with its primary role in mediating the agonist-induced release of arachidonic acid for eicosanoid production. It is subject to complex mechanisms of regulation that ensure that levels of free arachidonic acid are tightly controlled. The calcium-induced translocation of cPLA(2)alpha from the cytosol to membrane regulates its interaction with phospholipid substrate. cPLA(2)alpha is additionally regulated by phosphorylation on sites in the catalytic domain. Because of its central position as the upstream regulatory enzyme for initiating production of several classes of bioactive lipid mediators (leukotrienes, prostaglandins and platelet-activating factor), it is a potentially important pharmacological target for the control of inflammatory diseases.     

Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C

Inhibitors of phospholipase A2, tetracaine and quinacrine, inhibitors of protein kinases, H-7 and H-8, and a diacylglycerol lipase inhibitor reduced the level of CMV-induced [3H]AA release. A combination of H-7 and quinacrine inhibited stimulation of [3H]AA by about 80%. LU cells chronically treated with TPA and infected with CMV, had a reduced level of CMV-induced [3H]AA release and in the presence of quinacrine it was completely inhibited. These results suggest that CMV-induced stimulation of AA metabolism is mediated by pathways which are associated with activation of PLA2 and protein kinase C.     

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress-activated protein kinase pathways.

Stress-activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin-stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild-type human cPLA2 in vitro, although to different extents, but not cPLA2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42-kDa mitogen-activated protein kinase incorporated similar levels of phosphate into cPLA2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen-induced phosphorylation of cPLA2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially ( approximately 50%) blocks phosphorylation at both sites and to a similar extent in thrombin-stimulated platelets. Inhibition of phosphorylation resulted in a two- to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin- and collagen-stimulated platelets. Our data suggest that cPLA2 is a substrate for several SAPK cascades and that phosphorylation of cPLA2 augments arachidonic acid release.     

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.     

Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation.

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.     

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

Bradykinin (BK)-induced release of arachidonic acid (AA) fromMadin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol12-myristate 13-acetate (PMA) caused a synergistic increase in BK- andA-23187-induced release of AA but alone had no effect on this release.Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS)abolished the synergistic effects of PMA but did not affect AA releasecaused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMAresulted in a reduction of AA release induced by BK or A-23187addition, which corresponded to a decrease in cytoplasmic phospholipaseA₂(cPLA₂) activity as measured incell extracts. Although Western blotting revealed no differences in cPLA₂ expression as a result ofPMA treatment, phosphorylation of the enzyme, as assessed byphosphoserine content, was significantly reduced in PKC-depleted cells.These results imply that, with PKC downregulation, subsequent BKstimulation results in aCa²⁺-dependent translocation of aless phosphorylated, less active form ofcPLA₂. Any stimulation of PKC byBK addition did not appear as a significant event in onset reponsesleading to AA release. On the other hand, inhibition of themitogen-activated protein kinase (MAPK) cascade with the MAPK kinaseinhibitor, PD-98059, significantly decreased BK-induced release of AA,a finding that, with our other results, points to the existence of aPKC-independent route for stimulation of MAPK and the propagation ofonset responses.

     

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2.

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2-4 h) of arachidonic acid (AA) and prostaglandin (PG) D2 in rat liver macrophages. Group IV cytosolic phospholipase A2 (cPLA2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA2 shows an enhanced in vitro activity. The Ca2+ dependence of cPLA2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA2 mRNA (after 2-4 h) and an enhanced expression of cPLA2 protein (after 8 h). The cellular cPLA2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA2 (sPLA2). LPS has no effect on the levels of Groups V and IIA sPLA2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA2 protein and sPLA2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA2.     

Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.     

Amplification mechanisms of inflammation: paracrine stimulation of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid

In macrophages and other major immunoinflammatory cells, two phospholipase A(2) (PLA(2)) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A(2) (cPLA(2)) and a secreted phospholipase A(2) (sPLA(2)). In this study, the molecular mechanism underlying cross-talk between the two PLA(2)s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA(2)-dependent manner, and addition of exogenous group V sPLA(2) to the activated cells increases the release. This sPLA(2) effect is abolished if the cells are pretreated with cPLA(2) inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA(2). In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA(2) activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA(2) response to levels found in cells displaying normal cPLA(2) activity. Moreover, 12- and 15-HPETE are able to enhance sPLA(2) activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA(2)-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA(2) with cPLA(2) for a full AA mobilization response.     

The Bax N terminus is required for negative regulation by the mitogen-activated protein kinase kinase and Akt signaling pathways in T cells

The Bcl-2 family proapoptotic protein, Bax, redistributes to the mitochondrion in response to varied stimuli, triggering loss of mitochondrial integrity and apoptosis. Suppression of MAPK kinase (MEK1) by the reagent UO126 in activated T cells maintained in the cytokine IL-2 disrupts cytoplasmic localization of Bax and cell survival. UO126 triggers mitochondrial translocation of ectopically expressed Bax-GFP, and both UO126 and dominant negative MEK-1 (DN-MEK1) trigger increased apoptosis in Bax-GFP-expressing T cell lines. Because inhibition of PI3K or its target Akt also triggers mitochondrial translocation of Bax in T cells and apoptosis in Bax-transfected cell lines, we generated Bax deletion mutants to identify the region(s) that confers sensitivity to regulation by MEK1 and Akt. A deletion mutant (Bax(1-171)) without the C terminus mitochondrial targeting sequence or an Akt target site (Ser(184)) localizes to the cytoplasm and triggers low level apoptosis that is enhanced by DN-Akt or DN-MEK1. A construct that lacks the first 29 aa (Bax-delta29) largely localizes to mitochondria, is highly apoptogenic, and is not inhibited by Akt or MEK1. Furthermore, Bax-delta29 overcomes IL-2-dependent survival in a T cell line, whereas Bax triggers comparatively low levels of apoptosis in these cells. Cytoplasmic localization and regulation by MEK1 and Akt are restored in a mutant deleted of the first 13 aa (Bax-delta13). Taken together, our results identify a region in the Bax N terminus that determines cellular localization regulated by MEK- and Akt-dependent signaling in T cells.     

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.     

Cross-talk between cytosolic phospholipase A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release

Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IValpha cytosolic PLA2 (cPLA2alpha) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i). MC+/+, which lack group IIa and V PLA2s, and (ii). MC-/-, which lack groups IIa, V, and IValpha PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC-/-. It has been argued that cPLA2alpha plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IValpha PLA2s were expressed in MC-/- or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2alpha in H2O2-treated MC-/- increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC-/-. When sPLA2 and cPLA2alpha are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N',N'-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2alpha and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2alpha is responsible for AA release.     

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.     

Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate.

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.     

Role of secretory and cytosolic phospholipase A(2) enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA(2)) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA(2) enzymes and AA release was strongly reduced by cellular pretreatment with different PLA(2) inhibitors and by pertussis toxin, an inhibitor of G(i)-protein activation. This indicates that both cytosolic and secretory PLA(2) enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.     

Disruption of two putative nuclear localization sequences is required for cytosolic localization of mitogen-activated protein kinase phosphatase-2

MAP kinase phosphatase-2 (MKP-2) is a member of a family of dual specificity phosphatases (DSPs) that function in both the cytosol and nucleus to inactivate the MAP kinases. The mechanism that controls the subcellular distribution of these proteins is currently unclear. In this study, we have used site-directed mutagenesis to remove two novel nuclear localization sequences, NLS-1 and -2, either alone or in combination (DNLS). Loss of NLS-1 or NLS-2 alone did not alter the nuclear targeting of MKP-2 but mutation of both resulted in MKP-2 being retained within the cytosol. Furthermore, whilst expression of WT-MKP-2, NLS-1 or NLS-2 reduced both sorbitol- or UV-stimulated nuclear c-Jun N-terminal kinase (JNK) activity in HEK293 cells, this effect was absent in cells expressing DNLS-MKP-2. Similarly, transient transfection of WT-MKP-2, NLS-1 or NLS-2, but not DNLS-MKP-2 was able to substantially reduce agonist-stimulated ANF reporter activity in rat cardiac myocytes. Taken together, these results indicate that whilst both novel NLS participate in the nuclear localization of MKP-2, the expression of either sequence is sufficient to retain nuclear targeting.     

Inflammatory cytokines enhance muscarinic-mediated arachidonic acid release through p38 mitogen-activated protein kinase in A2058 cells

The human melanoma cell line A2058 expresses the Gq-coupled M5 subtype of muscarinic receptor. Stimulation with the cholinergic agonist, carbachol, induces a dose-dependent increase in arachidonic acid release. The carbachol-induced arachidonate release is potentiated two- to threefold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor-alpha or interleukin-1beta . Cytokine-induced enhancement of muscarinic-mediated arachidonic acid release peaks near 1 h. Western analysis suggests that both cytokines are capable of activating the nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK) pathways. Anisomycin (1 microM) treatment mimics the cytokine-induced enhancement of arachidonic acid production and activates the p38 MAPK pathway, but does not activate the NF-kappaB pathway. Furthermore, pre-treatment of A2058 cells with the putative p38 MAPK inhibitor, SB202190, ablates the cytokine-dependent augmentation without interfering with the muscarinic-mediated arachidonic acid release in untreated cells. Moreover, cytokine treatment does not affect other M5-coupled pathways (e.g., phospholipase C activity or intracellular Ca2+ mobilization), suggesting that p38 MAPK activation principally modulates muscarinic-mediated phospholipase A2 activity. Finally, in primary cultures of cells taken from rat cerebellum, key aspects of this finding are repeated in cultures enriched for glia, but not in cultures enriched for granule neurons.