1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Differential participation of phospholipase A(2) isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800μM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxigenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.     

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.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Localization and functional interrelationships among cytosolic Group IV, secreted Group V, and Ca2+-independent Group VI phospholipase A2s in P388D1 macrophages using GFP/RFP constructs

P388D(1) cells exposed to bacterial lipopolysaccharide (LPS) mobilize arachidonic acid (AA) for prostaglandin synthesis in two temporally distinct pathways. The "immediate pathway" is triggered within minutes by receptor agonists such as platelet-activating factor (PAF) but only if the cells have previously been primed with LPS for 1 h. The "delayed pathway" occurs in response to LPS alone over the course of several hours. We have now investigated the subcellular localization of both the Group IV cytosolic phospholipase A(2) (cPLA(2)) and the Group V secreted PLA(2) (sPLA(2)) during these two temporally distinct routes of AA release. We have prepared cells overexpressing fusion proteins of sPLA(2)-GFP and cPLA(2)-RFP. In the resting cells, cPLA(2)-RFP was uniformly located throughout the cytoplasm, and short-term treatment with LPS did not induce translocation to perinuclear and/or Golgi membranes. However, such a translocation occurred almost immediately after the addition of PAF to the cells. Long-term exposure of the cells to LPS led to the translocation of cPLA(2)-RFP to intracellular membranes after 3 h, and correlates with a significant release of AA in a cPLA(2)-dependent manner. At the same time period that the delayed association of cPLA(2) with perinuclear membranes is detected, an intense fluorescence arising from the sPLA(2)-GFP was found around the nucleus in the sPLA(2)-GFP stably transfected cells. In parallel with these changes, significant AA release was detected from the sPLA(2)-GFP transfectants in a cPLA(2)-dependent manner, which may reflect cross-talk between sPLA(2) and cPLA(2). The subcellular localization of the Group VIA Ca(2+)-independent PLA(2) (iPLA(2)) was also investigated. Cells overexpressing iPLA(2)-GFP showed no fluorescence changes under any activation condition. However, the iPLA(2)-GFP-expressing cells showed relatively high basal AA release, confirming a role for iPLA(2) in basal deacylation reactions. These new data illustrate the subcellular localization changes that accompany the distinct roles that each of the three kinds of PLA(2) present in P388D(1) macrophages play in AA mobilization.     

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.     

Effect of aging on the mechanisms of PTH-induced calcium influx in rat intestinal cells

We have investigated the effects of aging on parathyroid hormone (PTH) modulation of intracellular calcium homeostasis and their relationship to signal transduction pathways in isolated rat duodenal cells (enterocytes). PTH (10(-8)-10(-9) M) increased enterocyte (45)Ca(2+) influx and intracellular Ca(2+) concentration ([Ca(2+)](i)) to a greater extent (twofold and 50%, respectively) in aged (24 months) than in young (3 months) animals. The [Ca(2+)](i) response of old cells to the hormone was slower, lacking the early phase of changes in cytosolic Ca(2+). Ca(2+) influx induced by PTH was prevented by the protein kinase A antagonist Rp-cAMPS in both young and aged enterocytes, whereas neomycin and compound U73122, inhibitors of PLC-catalyzed phosphoinositide hydrolysis, abolished hormone-dependent Ca(2+) influx in young but had no effect on aged cells. Higher basal adenylyl cyclase (AC) activity and cAMP content were detected in old enterocytes. PTH increased the absolute levels of cAMP in aged cells and AC activity of microsomes isolated therefrom to a greater extent (>/= twofold) than in young enterocytes/membranes. In young cells, the hormone also induced a rapid and transient release of inositoltrisphosphate (IP(3)) and diacylglycerol (neomycin-sensitive) at 45 sec, and a delayed phase of DAG at 5 min (neomycin-insensitive). The early formation of IP(3) and DAG was blunted in aged animals. These results suggest that both the PLC and adenylyl cyclase cascades are involved in PTH stimulation of Ca(2+) influx in duodenal cells. During aging, however, only the cAMP pathway is operative, mediating a potentiation of the effects of the hormone. Additional studies are required to establish the relative role of PTH-dependent messenger systems in the regulation of intestinal calcium absorption and age-related abnormalities.     

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).     

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.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Phospholipase A(2) isoforms: a perspective

Several new PLA(2)s have been identified based on their nucleotide gene sequences. They were classified mainly into three groups: cytosolic PLA(2) (cPLA(2)), secretary PLA(2) (sPLA(2)), and intracellular PLA(2) (iPLA(2)). They differ from each other in terms of substrate specificity, Ca(2+) requirement and lipid modification. The questions that still remain to be addressed are the subcellular localization and differential regulation of the isoforms in various cell types and under different physiological conditions. It is required to identify the downstream events that occur upon PLA(2) activation, particularly target protein or metabolic pathway for liberated arachidonic acid or other fatty acids. Understanding the same will greatly help in the development of potent and specific pharmacological modulators that can be used for basic research and clinical applications.The information of the human and other genomes of PLA(2)s, combined with the use of proteomics and genetically manipulated mouse models of different diseases, will illuminate us about the specific and potentially overlapping roles of individual phospholipases as mediators of physiological and pathological processes. Hopefully, such understanding will enable the development of specific agents aimed at decreasing the potential contribution of individual secretary phospholipases to vascular diseases.The signaling cascades involved in the activation of cPLA(2) by mitogen activated protein kinases (MAPKs) is now evident. It has been demonstrated that p44 MAPK phosphorylates cPLA(2) and increases its activity in cells and tissues. The phosphorylation of cPLA(2) at ser505 occurs before the increase in intracellular Ca(2+) that facilitate the binding of the lipid binding domain of cPLA(2) to phospholipids, promoting its translocation to cellular membranes and AA release. Recently, a negative feed back loop for cPLA(2) activation by MAPK has been proposed. If PLA(2) activation in a given model depends on PKC, PKA, cAMP, or MAPK then inhibition of these phosphorylating enzymes may alter activities of PLA(2) isoforms during cellular injury. Understanding the signaling pathways involved in the activation/deactivation of PLA(2) during cellular injury will point to key events that can be used to prevent the cellular injury. Furthermore, to date, there is limited information available regarding the regulation of iPLA(2) or sPLA(2) by these pathways.     

Role of Ca2+-independent phospholipase A2 on arachidonic acid release induced by reactive oxygen species

Previous studies have shown that reactive oxygen species (ROS) enhance arachidonic acid (AA) release and the subsequent AA metabolism in macrophages. The purpose of this study was determined the implication of phospholipases A2 (PLA2s) in these events. Our results show that oxidative stress induced by exogenous adding of hydrogen peroxide or superoxide anion in macrophage RAW 264.7 and mouse peritoneal macrophage cultures caused a marked enhancement of calcium-independent PLA2 (iPLA2) activity,whereas the increment of secreted PLA2 (sPLA2) and calcium-dependent cytosolic PLA2 (cPLA2) activities were slight. This increase of iPLA2 activity by ROS was rapid and dose-dependent. ROS also induced a significant [3H] arachidonic acid (AA) release. The iPLA2 selective inhibitor, bromoenol lactone, almost completely suppressed the mobilization of [3H]AA induced by ROS whereas antisense oligonucleotide against cPLA2 did not have any appreciable effect. Thus, our data show that iPLA2 activity is involved in the mechanism by which ROS increases the availability of free AA in macrophages RAW 264.7. Moreover, the protein kinase C (PKC) inhibitor, calphostin C, and calcium chelators had no effect on the [3H]AA release induced by ROS, suggesting this is a regulatory role of iPLA2.     

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)β, to lipid mobilization in proximal mast cells and distal fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.     

Phospolipase A2 and apoptosis

Phospolipase A(2) (PLA(2)) is the esterase activity that cleaves the sn-2 ester bond in glycerophospholipids, releasing free fatty acids and lysophospholipids. The PLA(2) activity is found in a variety of enzymes which can be divided in several types based on their Ca(2+) dependence for their activity; Ca(2+)-dependent secretory phosholipases (sPLA(2)s) and cytosolic phospholipases (cPLA(2)s), and Ca(2+)-independent phospholipase A(2)s (iPLA(2)s). These enzymes also show diverse size and substrate specificity (i.e., in the fatty acid chain length and extent of saturation). Among the fatty acids released by PLA(2), arachidonic acid (AA) is of particular biological importance, because it is subsequently converted to prostanoids and leukotrienes by cyclooxygenases (COX) and lipoxygenases (LOX), respectively. Free AA may also stimulate apoptosis through activation of sphingomyelinase. Alternatively, it is suggested that oxidized metabolites generated from AA by LOX induce apoptosis. Although the precise mechanisms remain to be elucidated, changes are observed in glycerolipid metabolism during apoptotic processes. In some cells induced to undergo apoptosis, AA is released concomitant with loss of cell viability, caspase activation and DNA fragmentation. Such AA releases appear to be mediated by activation of cPLA(2) and/or iPLA(2). For example, tumor necrosis factor-alpha (TNF-alpha)-induced cell death is mediated by cPLA(2), whereas Fas-induced apoptosis appears to be mediated by iPLA(2). Some discrepancies among early experimental results were probably caused by differences in the experimental conditions such as the serum concentration, inhibitors used that are not necessarily specific to a single-type enzyme, or differential expression of each PLA(2) in cells employed in the experiments. Recent studies eliminated such problems, by carefully defining the experimental conditions, and using multiple inhibitors that show different specificities. Accordingly, more convincing data are available that demonstrate involvement of some PLA(2)s in the apoptotic processes. In addition to cPLA(2) and iPLA(2), sPLA(2)s were recently found to play roles in apoptosis. Moreover, new proteins that appear to control PLA(2)s are being discovered. Here, the roles of PLA(2)s in apoptosis are discussed by reviewing recent reports.     

Hydrogen peroxide activation of Ca(2+)-independent phospholipase A(2) in uterine stromal cells

In rat uterine stromal cells (U(III) cells), an oxidative stress induced by H(2)O(2) caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca(2+) concentration and was not inhibited by Ca(2+)-dependent phospholipase A(2) (cPLA(2)) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H(2)O(2) treatment did not impair AA esterification but significantly increased Ca(2+)-independent PLA(2) (iPLA(2)) activity. Since iPLA(2) specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H(2)O(2), we conclude that iPLA(2) activity represents the major mechanism by which H(2)O(2) increases the availability of non-esterified AA in U(III) cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H(2)O(2), suggesting a regulatory mechanism of iPLA(2) by PKC that remains to be clarified.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.