A gene (pks2) encoding a putative 6-methylsalicylic acid synthase from Glarea lozoyensis.

A gene that encodes for a polyketide synthase (PKS) was cloned from the fungus Glarea lozoyensis and characterized. The gene (pks2) consists of four exons interrupted by three introns of 51, 59, and 65 bp, which are clustered at the 5' end. Its predicted product is a 1791-amino-acid protein containing five catalytic motifs typical of fungal PKSs, including a beta-ketosynthase, an acyltransferase, a dehydratase, a beta-ketoacyl reductase, and an acyl carrier region. The gene is transcribed from an initiation site located 375 bp upstream of the translational start codon and extends to a transcriptional termination site 244 bp downstream of the translational stop codon. The gene function is not required for either vegetative growth of G. lozoyensis or for production of pneumocandin, as shown by Agrobacterium-mediated pks2 gene disruption. Previously reported cluster analysis of ketosynthase motifs from 37 fungal polyketide synthases had grouped the Pks2p from G. lozoyensis with PKSs involved in the biosynthesis of 6-methylsalicylic acid. To verify the function of the gene, it was transferred into Aspergillus nidulans under the control of the trpC promoter. 5'-and 3'-RACE experiments confirmed that it was transcribed in the heterologous host, and was associated with the synthesis of a compound identified as 6-methylsalicylic acid by NMR and mass spectrometry. In G. lozoyensis, pks2 is flanked by a gene that encodes a putative drug resistance efflux pump. The Aspergillus pks2 transformants, which were arginine prototrophs, also exhibited precocious pigmentation and accumulated a benzophenone that appeared to be a precursor of emericellin (variecoxanthone B), a known product of A. nidulans. The buildup of the benzophenone may be related to the use of an alternative splice site for the removal of intron 1 of the pks2 transcript in the heterologous host.

Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.

Glarea lozoyensis produces pneumocandin B(0), a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)(2)Cys(6) binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.

Differential effects of SB 242235, a selective p38 mitogen-activated protein kinase inhibitor, on IL-1 treated bovine and human cartilage/chondrocyte cultures.

The p38 MAP kinase inhibitor, SB 242235, was evaluated for its effects on the metabolism of bovine and human cartilage and primary chondrocyte cultures. SB 242235 had no effect on proteoglycan synthesis (PG) in bovine articular cartilage explants (BAC), as measured by [(35)S]-sulfate incorporation into glycosaminoglycans (GAGs). In addition, the compound had no effect on IL-1 alpha-induced GAG release from these cultures. However, there was a potent, dose-dependent inhibition of nitric oxide (NO) release from IL-1 alpha-stimulated BAC with an IC(50)of approximately 0.6 microM, with similar effects observed in primary chondrocytes. The effect on BAC was time dependent, and mechanistically did not appear to be the result of inhibition of protein kinase C (PKC), protein kinase A (PKA) or MEK-1. The effect on NO release in bovine chondrocytes was at the level of inducible nitric oxide synthase (iNOS) gene expression, which was inhibited at similar concentrations as nitrite production. In primary human chondrocytes, IL-1 beta induction of p38 MAP kinase was inhibited by SB 242235 with an IC(50)of approximately 1 microM. Surprisingly, however, treatment of IL-beta-stimulated human cartilage or chondrocytes with SB 242235 did not inhibit either NO production or the induction of iNOS. On the other hand, the natural product hymenialdisine (HYM), a protein tyrosine kinase (PTK) inhibitor, inhibited NO production and iNOS in both species. In contrast to the differential control of iNOS, PGE(2)was inhibited by SB 242235 in both IL-1-stimulated bovine and human chondrocyte cultures. These studies indicate that there are species differences in the control of iNOS by p38 inhibitors and also that different pathways may control IL-1-induced proteoglycan breakdown and NO production.

Modulation of human monocyte activities by tranilast, SB 252218, a compound demonstrating efficacy in restenosis.

Tranilast (SB 252218) is a compound initially identified as an anti-atopic agent. Recently the compound has demonstrated clear beneficial effects in animal models of restenosis. Here we confirm tranilast has broad and profound effects on human monocytes, which could contribute to the vascular antifibrotic activity. Tranilast exhibited significant immunomodulatory activity inhibiting endotoxin-induced prostaglandin E(2) (PGE(2); IC(50) = approximately 1-20 microM), thromboxane B(2) (IC(50) = approximately 10-50 microM), transforming growth factor-beta1 (TGF-beta1; IC(50) = approximately 100-200 microM), and interleukin-8 (IC(50) = approximately 100 microM) formation, but had no effect on tumor necrosis factor-alpha. Interleukin-12 and -18-induced interferon-gamma formation by monocytes was also attenuated by tranilast. A23187-induced monocyte leukotriene C(4) or PGE(2) formation was inhibited by tranilast at IC(50) values of 10-40 microM and 2-20 microM, respectively, incubated with or without exogenous arachidonic acid. Interestingly, tranilast (up to 1000 microM) had no direct effects on cyclooxygenase I or II activity, nor did it have significant effects on human type IIA 14 kDa or type IV 85 kDa phospholipase A(2) activity. Furthermore, tranilast had no effect on endotoxin-induced cyclooxygenase II protein expression, suggesting tranilast modulates eicosanoid production and release by an as yet unidentified mechanism. Alternatively, the expression of TGF-beta1 was inhibited by tranilast but found to be due in part to inhibition of PGE(2) because exogenous PGE(2) could abrogate tranilast-mediated inhibition of TGF-beta1. Taken together, although a reported direct inhibitor of fibroblast proliferation, we show tranilast also attenuates the proinflammatory activity of human monocytes, adding to its potential efficacy as a therapeutic agent in restenosis.

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation.

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.

Decreased nuclear factor-kappaB DNA binding activity following permanent focal cerebral ischaemia in the rat.

Many factors implicated in the pathogenesis of cerebral ischaemia such as glutamate, tumour necrosis factor and interleukin-1 have also been shown to activate nuclear factor-kappaB (NF-kappaB). In the present study we have investigated NF-kappaB activity at various times following permanent focal cerebral ischaemia in rats using immunohistochemistry, western blotting and electrophoretic mobility shift assay (EMSA). Three hours following middle cerebral artery occlusion nuclear translocation of NF-kappaB was detected using immunohistochemical and western blotting techniques. This was reflected in a trend towards increased NF-kappaB binding activity (EMSA) in the ischaemic cortex compared to histologically normal tissue. In contrast however, from 6 to 48 h post-occlusion nuclear translocation and NF-kappaB binding activity was decreased in the ischaemic cortex. Decreased NF-kappaB binding activity detected in degenerating neurones, suggests that decreased NF-kappaB activity may exacerbate ischaemia induced neuronal cell death.

An indolocarbazole inhibitor of human checkpoint kinase (Chk1) abrogates cell cycle arrest caused by DNA damage.

Many cancer therapies cause DNA damage to effectively kill proliferating tumor cells; however, a major limitation of current therapies is the emergence of resistant tumors following initial treatment. Cell cycle checkpoints are involved in the response to DNA damage and specifically prevent cell cycle progression to allow DNA repair. Tumor cells can take advantage of the G2 checkpoint to arrest following DNA damage and avoid immediate cell death. This can contribute to acquisition of drug resistance. By abrogating the G2 checkpoint arrest, it may be possible to synergistically augment tumor cell death induced by DNA damage and circumvent resistance. This requires an understanding of the molecules involved in regulating the checkpoints. Human Chk1 is a recently identified homologue of the Schizosaccharomyces pombe checkpoint kinase gene, which is required for G2 arrest in response to DNA damage. Chk1 phosphorylates the dual specificity phosphatase cdc25C on Ser-216, and this may be involved in preventing cdc25 from activating cdc2/cyclinB and initiating mitosis. To further study the role of Chk1 in G2 checkpoint control, we identified a potent and selective indolocarbazole inhibitor (SB-218078) of Chk1 kinase activity and used this compound to assess cell cycle checkpoint responses. Limited DNA damage induced by gamma-irradiation or the topoisomerase I inhibitor topotecan was used to induce G2 arrest in HeLa cells. In the presence of the Chk1 inhibitor, the cells did not arrest following gamma-irradiation or treatment with topotecan, but continued into mitosis. Abrogation of the damage-arrest checkpoint also enhanced the cytotoxicity of topoisomerase I inhibitors. These studies suggest that Chk1 activity is required for G2 arrest following DNA damage.

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase.

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.

Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

Respective roles of the 14 kDa and 85 kDa phospholipase A2 enzymes in human monocyte eicosanoid formation.

Human monocytes possess both the cytosolic 85 kDa phospholipase (PLA) A2 and a 14 kDa PLA2 and are capable of simultaneously producing prostanoids (PG), leukotrienes (LT) and platelet activating factor (PAF). As the exact roles of the two enzymes in monocyte lipid mediator formation was unclear, both selective PLA2 inhibitors and antisense were used to elucidate their respective roles. Reduction in 85 kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85 kDa PLA2 inhibitor, arachidonyl trifluormethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocytes prostanoid formation but not LTC4 or PAF production. This confirmed the important role of the 85 kDa PLA2 in prostanoid formation but indicated a less significant role in LT or PAF biosynthesis. Alternatively, treatment of monocytes with the selective, active-site-directed 14 kDa PLA2 inhibitor, SB 203347, totally inhibited LT and PAF formation, while prostanoid formation was not altered. Addition of 20 uM exogenous arachidonic acid (AA) to monocytes exposed to SB 203347 did not alter A23187-induced LTC4 generation, indicating that SB 203347 had no effect on downstream AA metabolizing enzymes in this setting. Taken together, these results provide evidence that the 14 kDa PLA2 provides substrate for monocyte LT and PAF formation, while the 85 kDa PLA2 plays a more significant role in the formation of PG.

Cytosolic 85-kDa phospholipase A2-mediated release of arachidonic acid is critical for proliferation of vascular smooth muscle cells.

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2 activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 mu M) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2 (PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.

Inhibition of NFkappaB-mediated interleukin-1beta-stimulated prostaglandin E2 formation by the marine natural product hymenialdisine.

Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM). Alternatively, both an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, were without affect. Direct action of hymenialdisine on IL-1-induced NFkappaB activation was demonstrated by a significant reduction (approximately 80%) in NFkappaB binding to the classical kappaB consensus motif (as assessed by electrophoretic mobility shift assay) and inhibition of stimulated p65 migration from the cytosol of treated cells (as assessed by Western analysis). Consistent with the role of NFkappaB in the transcriptional regulation of COX II and 85-kDa PLA2, hymenialdisine-treated RSF did not transcribe the respective mRNAs in response to IL-1. This led to reductions in their respective protein levels and subsequent reductions in the ability to produce PGE2. Specificity of action is suggested as IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFkappaB-regulated event, was also inhibited by hymenialdisine, whereas IL-1-induced production of vascular endothelial growth factor, a non-NFkappaB-regulated gene, was not affected by exposure to hymenialdisine. Taken together, hymenialdisine inhibits IL-1-stimulated-RSF PGE2 formation acting predominately through modulation of NFkappaB activation and offers an interesting novel tool to evaluate the role of NFkappaB in inflammatory disease.

Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes.

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

Depletion of human monocyte 85-kDa phospholipase A2 does not alter leukotriene formation.

Human monocytes possess several acylhydrolase activities and are capable of producing both prostanoids (PG) and leukotriene (LT) products upon acute stimulation with calcium ionophore, A23187 or phagocytosis of zymosan particles. The cytosolic 85-kDa phospholipase (PLA) A2 co-exists with the 14-kDa PLA2 in the human monocyte, but their respective roles in LT production are not well understood. Reduction in 85-kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85-kDa PLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocyte prostanoid formation. In contrast, neither treatment altered stimulated LTC4 production. This confirmed the important role of the 85-kDa PLA2 in prostanoid formation but suggests that it has less of a role in LT biosynthesis. Alternatively, treatment of monocytes with the selective, active site-directed 14-kDa PLA2 inhibitor, SB 203347, prior to stimulation had no effect on prostanoid formation at concentrations that totally inhibited LT formation. Addition of 20 microM exogenous arachidonic acid to monocytes exposed to SK 7111 or SB 203347 did not alter A23187-induced PGE2 or LTC4 generation, respectively, indicating that these agents had no effect on downstream arachidonic acid-metabolizing enzymes in this setting. Taken together, these results provide evidence that the 85-kDa PLA2 may play a more significant role in the formation of PG than LT. Further, utilization of SB 203347 provides intriguing data to form the hypothesis that a non-85-kDa PLA2 sn-2 acyl hydrolase, possibly the 14-kDa PLA2, may provide substrate for LT formation.

Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

Suppression of human synovial fibroblast 85 kDa phospholipase A2 by antisense reduces interleukin-1 beta induced prostaglandin E2.

OBJECTIVE: To evaluate the relative roles of rheumatoid synovial fibroblast phospholipases A2 (PLA2) in interleukin- 1beta (IL-1 beta) stimulated prostaglandin E2 (PGE2) production. METHODS: The role of the cytosolic 85 kDa PLA2 in IL-1beta induced human rheumatoid synovial fibroblast PGE2 formation was directly evaluated using an antisense phosphorothioate oligonucleotide to the initiation site of the 85 kDa PLA2 mRNA. Contribution of the 14 kDa PLA2 was assessed using selective inhibitors or a neutralizing monoclonal antibody (Mab). RESULTS: Antisense, but not sense, decreased IL-1beta upregulation of 85 kDa PLA2 activity and protein levels. The antisense effect was specific, since it did not affect 14 kDa PLA2 activity released into the media or induced cyclooxygenase II protein levels over 24 h. Antisense, but not sense, reduced PGE2 formation in a concentration dependent manner. IL-1 beta significantly upregulated cell associated 14 kDa PLA2 and its subsequent release. Specific inhibition of this enzyme by a neutralizing Mab or selective inhibitors of 14 kDA PLA2 activity did not alter IL-1beta induced PGE2 levels. CONCLUSION: These data directly support a role for the 85 kDa PLA2, but not the 14 kDa PLA2, in IL- 1beta stimulated PGE2 production from human rheumatoid synovial fibroblast.

SB 203347, an inhibitor of 14 kDa phospholipase A2, alters human neutrophil arachidonic acid release and metabolism and prolongs survival in murine endotoxin shock.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.5 microM) but exhibits a 40-fold weaker inhibition of 85 kDa PLA2 (IC50 = 20 microM) using [3H]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA2 was confirmed both by observing the pH dependence of its IC50 and by demonstrating linear inhibition in a "scooting" kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating the effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA2 activity in acid extracted-intact human neutrophil homogenate (IC50 = 4.7 microM). SB 203347 inhibited A23187-induced intact human neutrophil AA mass release in a concentration-dependent manner (IC50 = 1 microM), which coincided with reductions in the biosynthesis of platelet-activating factor (IC50 = 1.5 microM) and leukotriene B4 (IC50 = 2.3 microM). Finally, SB 203347 prolonged survival in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA2 in the formation of AA-derived proinflammatory lipid mediator.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of monocyte 85-kDa phospholipase A2 by antisense and effects on endotoxin-induced prostaglandin biosynthesis.

Studies were conducted to characterize a human monocyte model where the role of the 85-kDa phospholipase A2 (PLA2) in prostanoid formation could be evaluated. The presence of an immunologically related 85-kDa PLA2 and type II 14-kDa PLA2 was demonstrated in human monocytes and their roles examined in lipopolysaccharide (LPS)-induced monocyte prostaglandin E2 (PGE2) formation. Exposure of human monocytes to LPS over 18 h resulted in the up-regulation of the mitogen-inducible cyclooxygenase-2 and was accompanied by production and release of prostaglandin E2 but not leukotriene C4. This coincided with a 2-fold increase in the 85-kDa PLA2 protein and activity levels. In contrast, there was no effect on the type II 14-kDa-like PLA2 activity measured in the 100,000 x g particulate fraction nor did LPS induce the release of type II 14-kDa PLA2 into the medium. Treatment with cycloheximide over 18 h resulted in a time-dependent decrease in cytosolic 85-kDa PLA2 protein and activity (half-life = 4 h), but there was no change in the particulate type II 14-kDa-like PLA2 activity. Monocytes were therefore exposed to an 85-kDa PLA2 initiation site-directed antisense oligonucleotide which specifically decreased the cytosolic 85-kDa PLA2 protein levels and activity in a concentration-dependent manner. This had no effect on the cyclooxygenase-2 (protein mass or the ability to convert arachidonic acid to PGE2) or the particulate fraction sn-2 acylhydrolytic activity but was associated with a decrease in LPS-induced PGE2 production. Taken together, these data support a role for the cytosolic 85-kDa PLA2 in LPS-induced monocyte PGE2 formation.