Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes

In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.     

Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187.     

The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift.

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.     

Cytosolic phospholipase A2 is required for optimal ATP activation of BK channels in GH(3) cells

To test the hypothesis that ATP activation of BK channels in GH(3) cells involves cytosolic phospholipase A(2) (cPLA(2)) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA(2) by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA(2), phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA(2)-alpha (but not cPLA(2)-gamma) as one target protein for phosphorylation that is intimately associated with the BK channel protein.     

Endothelial ICAM-1 protein induction is regulated by cytosolic phospholipase A2α via both NF-κB and CREB transcription factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.     

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.     

Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein

We sought to determine the roles of PKCalpha and G(i)alpha in regulating cPLA(2) activity in bovine pulmonary artery endothelial cell membrane under peroxynitrite (ONOO(-)) stimulation. Treatment of bovine pulmonary artery endothelial cells with ONOO(-) markedly stimulates the cell membrane associated protease activity, protein kinase C (PKC) activity, phospholipase A(2) (PLA(2)) activity, and arachidonic acid (AA) release from the cells. ONOO(-) significantly increases (Ca(2+))(i) in the cells, and pretreatment with the intracellular Ca(2+) chelator BAPTA-AM prevents the increase in (Ca(2+))(i), protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF(3)) (a cPLA(2) inhibitor) prevents ONOO(-)-stimulated cPLA(2) activity and AA release without producing a significant alteration of the protease activity. Pretreatment with vitamin E and aprotinin prevents ONOO(-)-induced increase in the protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment with the PKC inhibitor calphostin C prevents ONOO(-)-caused increase in PKC activity and cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot study of the cell membrane isolated from the ONOO(-)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. An immunoblot study with anti-nitrotyrosine antibody revealed that ONOO(-) induces nitration of tyrosine residues in PKCalpha. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. An immunoblot study of the endothelial cell membrane with polyclonal cPLA(2) antibody revealed that treatment of the cells with ONOO(-) markedly increases the cPLA(2) immunoreactive protein profile in the membrane. Pretreatment of the endothelial cells with Go6976, a PKCalpha inhibitor, prevents the increase in PKC activity and cPLA(2) activity in the cell membrane under ONOO(-)-triggered condition. It, therefore, appears from the present study that treatment of the cells with ONOO(-) causes an increase in the protease activity, and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot assay with polyclonal G(i)alpha antibody elicited an immunoreactive band having a molecular mass of 41 kDa. Pretreatment of the cells with pertussis toxin markedly inhibits ONOO(-)-induced increase in cPLA(2) activity and AA release without significantly altering (Ca(2+))(i), protease activity, and PKC activity in the cell membrane. Treatment of the cells with ONOO(-) causes phosphorylation of G(i)alpha in the cell membrane, and pretreatment with Go6976 prevents its phosphorylation. We suggest the existence of a pertusssis toxin sensitive G protein-mediated mechanism for activation of cPLA(2) by ONOO(-) in bovine pulmonary artery endothelial cell membrane, which is regulated by PKCalpha-dependent phosphorylation and sensitive to aprotinin for its inhibition.     

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated.In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.     

Activation of cytosolic phospholipase A2alpha through nitric oxide-induced S-nitrosylation. Involvement of inducible nitric-oxide synthase and cyclooxygenase-2

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)alpha protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)alpha activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.     

Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2)

Calcium-/calmodulin-dependent protein kinase II (CaM kinase II), a decoder of Ca(2+) signals, and cytosolic phospholipase A(2) (cPLA(2)), an enzyme involved in arachidonate release, are involved in many physiological and pathophysiological processes. Activation of CaM kinase II in norepinephrine-stimulated vascular smooth muscle cells leads to activation of cPLA(2) and arachidonic acid release. Surface plasmon resonance, mass spectrometry, and kinetic studies show that CaM kinase II binds to cPLA(2) resulting in cPLA(2) phosphorylation on Ser-515 and an increase in its enzymatic activity. Phosphopeptide mapping studies with cPLA(2) from norepinephrine-stimulated smooth muscle cells indicates that phosphorylation of cPLA(2) on Ser-515, but not on Ser-505 or Ser-727, occurs in vivo. This novel signaling pathway for arachidonate release is shown to be cPLA(2)-dependent by use of a recently described and highly selective inhibitor of this enzyme.     

Cytosolic phospholipase A(2) activity associated with nuclei is not inhibited by arachidonyl trifluoromethyl ketone in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin

We have studied the translocation of cytosolic phospholipase A(2) (cPLA(2)) to nuclei in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*). Translocation of phosphorylated cPLA(2) to nuclei was determined by immunoprecipitation of cPLA(2) in (32)P(i)-labeled cells. The identity of cPLA(2) was established by comparing its mobility on gels with an authentic cPLA(2) standard. cPLA(2) activity was quantified by measuring the release of [(14)C]arachidonic acid from the substrate 1-palmitoyl-2-[1-(14)C]arachidonyl-sn-glycerophosphatidylcholine. alpha(2)M* caused a two- to threefold increase in cPLA(2) phosphorylation and its translocation to nuclei. The p38 MAPK inhibitor SB203580, PKC inhibitor chelerythrin, or depletion of intracellular Ca(2+) profoundly decreased cPLA(2) activity in nuclei isolated from agonist-stimulated cells. The requirement for Ca(2+), PKC, and p38 MAPK activation appears to be of major importance for nuclear cPLA(2) activity. In contrast to cellular cPLA(2) activity, nuclear cPLA(2) activity was not inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)) in agonist-stimulated cells. It is concluded that the association of cPLA(2) with nuclear membranes in agonist-stimulated cells modifies the activity and the sensitivity of the enzyme to inhibition by AACOCF(3) in this phospholipid environment.     

Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown

This report documents the characterization of a novel mouse oocyte protein which was originally identified by microsequence analysis of a 67.8 kDa protein spot (pI 5.7) on a Coomassie-stained two-dimensional (2D) gel of murine egg proteins. Tandem mass spectroscopic analysis of the peptides obtained from the cored protein yielded sequences that appeared to match only ovary, egg, and preimplantation embryo cDNAs. We then cloned the novel gene by RACE-PCR, and analysis of the deduced cDNA sequence found that this maternal product was ∼56% identical to human cytosolic phospholipase A2γ (cPLA2γ). Based on this sequence homology, we named the molecule mouse cytosolic phospholipase A2γ (cPLA2γ). As with human cPLA2γ, mouse cPLA2γ contains a lipase consensus sequence and lacks the calcium binding domain that is found in other PLA2 proteins. However, mouse cPLA2γ is different from human cPLA2γ in that mouse cPLA2γ expression is restricted to the ovary and that the protein does not contain the myristoylation and prenylation lipid-anchoring motifs that are present in human cPLA2γ. Within oocytes, mouse cPLA2γ localizes mainly to the oocyte cortex and to the nucleoplasm. Interestingly, during germinal vesicle breakdown, mouse cPLA2γ aggregates dynamically relocate from the oocyte cortex to the nuclear envelope, suggesting a possible role for this putative egg-restricted phospholipase A2γ in membrane remodeling. Furthermore, mouse cPLA2γ protein continues to be expressed in the embryo until the 4-8-cell stage of development, suggesting that mouse cPLA2γ may function as a previously uncharacterized maternal effect gene.     

A VE-cadherin-PAR3-α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A(2)α in endothelial cells

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not β-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.     

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).     

The confluence-dependent interaction of cytosolic phospholipase A2-alpha with annexin A1 regulates endothelial cell prostaglandin E2 generation

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.     

Cloning and expression of the gene encoding phospholipase A1 from Serratia sp. MK1 in Escherichia coli.

The gene encoding extracellular phospholipase A1 of Serratia sp. MK1 was cloned from a genomic DNA library. Formation of transparent halos on the PCY agar plates was used to identify E. coli carrying the phospholipase A1 gene. A 4.2 kb EcoRI fragment was isolated and sequenced. From nucleotide sequences and expression of various plasmids, two open reading frames (plaA and plaS) involved in efficient expression of phospholipase A1 in natural and recombinant host were identified. Extracellular phospholipase A1 activity was identified as the gene product of plaA encoding 321 amino acids with a predicted MW of 33,400. Analysis of the amino acid sequence revealed significant homology (around 70%) to phospholipase A1 of Serratia liquefaciens and Yersinia enterocolitica. The sequence, -Gly-X1-Ser-X2-Gly-, known as a lipase-specific consensus sequence was also found in the bacterial phospholipase A1. PlaS encoding a protein of 224 amino acids showed no enzymatic activity, but might be necessary for the efficient expression of phospholipase A1 in E. coli. To further improve the production of phospholipase A1 as a soluble and active form in E. coli, the effect of some parameters was examined. Surprisingly, a higher yield of soluble and active phospholipase A1 could be obtained under the combined conditions of a lower temperature, an enriched medium, and a lower-strength promoter.     

The mediating role of cPLA2 in IL-1 beta and IL-6 release in LPS-induced HeLa cells

Studies were conducted to characterize a HeLa cell model by which the roles of the 85-kDa phospholipase A2 (cPLA2) in interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) release could be evaluated. At first, untreated HeLa cells were compared with lipopolysaccharide (LPS)-treated HeLa cells. The latter resulted in cPLA2 overexpression and an increased trend of IL-1 beta and IL-6 release. The indicated doses of 85-kDa cPLA2 antisense oligonucleotide directed against the initiation site were then used to block cPLA2 in LPS-induced HeLa cells. The process led to a dose-dependent decrease in cPLA2 protein with no noticeable change of cPLA2 mRNA. Compared with that of LPS added only, a reduction of IL-1 beta and IL-6 levels in the supernatants of transfected cells following the repression of cPLA2 was observed. These results suggested that 85-kDa cPLA2 may mediate the signalling cascades by which IL-1 beta and IL-6 were released in LPS-induced HeLa cells.