Phosphatidylcholine-specific Phospholipase C Inhibitor Research Articles

Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats

Recently, we reported that intracerebroventricularly (i.c.v.) administered histamine evokes the secretion of noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats. These results suggest the involvement of brain arachidonic acid cascade in the histamine-induced activation of the central adrenomedullary outflow. Arachidonic acid is released mainly by phospholipase A2 (PLA2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, histamine (27 nmol/animal, i.c.v.) -induced elevation of plasma noradrenaline and adrenaline was dose-dependently reduced by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.), ET-18-OCH3 (phosphatidylinositol-specific PLC inhibitor) (10 and 30 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 micromol/animal, i.c.v.). However, mepacrine (PLA2 inhibitor) (1.1 and 2.2 micromol/animal, i.c.v.) and D609 (phosphatidylcholine-specific PLC inhibitor) (30, 100 and 300 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain phosphatidylinositol-specific PLC and diacylglycerol lipase in the centrally administered histamine-induced activation of the adrenomedullary outflow in rats.

Silica Induces Macrophage Cytokines through Phosphatidylcholine-Specific Phospholipase C with Hydrogen Peroxide

Silica particle-associated inflammation is implicated in the genesis of several pulmonary diseases, including silicosis and lung cancer. In this study we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in silica-stimulated induction of TNF-alpha and IL-1beta and how PC-PLC activity is regulated by silica in a rat alveolar macrophage model. We demonstrated that inhibition of PC-PLC, which was achieved with tricychodecan-9-yl-xanthate (D609), blocked the silica-stimulated induction of TNF-alpha and IL-1beta in alveolar macrophage, suggesting that PC-PLC is involved in the silica-associated inflammatory response. PC-PLC activity was increased significantly by silica exposure, and this could be inhibited by MnTBAP, which catalyzes both the dismutation of O2.- to O2 and H2O2 and the dismutation of H2O2 to O2 and H2O, revealing that PC-PLC activity is regulated in a redox-dependent manner. This is further confirmed by the finding that PC-PLC activity was increased by exogenous H2O2. The intracellular calcium chelator BAPTA blocked the H2O2-increased PC-PLC activity, while the calcium ionophore, A23187, enhanced PC-PLC activity. The data indicate that PC-PLC plays critical roles in the silica-associated inflammatory response and that PC-PLC is regulated through redox- and calcium-dependent manners in alveolar macrophages.

Inhibition of PC-PLC Blocked the Survival of Mouse Neural Cells by Up-Regulating the Expression of Integrin beta4 and Rb

In order to understand the survival signals of neural cells during the development of the central nervous system (CNS), we explored the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in the survival of primary culture mouse neural cells (MNCs). We found that when the activity of PC-PLC in the neural cells was suppressed by its specific inhibitor, D609 (tricyclodecan-9-yl-xanthogenate), the cell survival was blocked, the viability of the cells remarkably declined. At the same time, the expressions of integrin beta4 and Rb protein were evidently elevated. We also examined the intracellular ROS (reactive oxygen species) levels after the cells were treated with D609. The result showed that the ROS level in neural cells was reduced. Our data suggested that PC-PLC had an important role in regulating neural cell survival, and PC-PLC might perform its function by upregulating the expressions of integrin beta4 and Rb protein. The changes of intracellular ROS level induced by D609 indicated that a modest level of ROS might be indispensable to neural cell survival.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-zeta activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-zeta, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-zeta and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.

Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca(2+) levels ([Ca(2+)](i)). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A(2) (cPLA(2)) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca(2+)](i) and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca(2+) in the culture media, D609 completely prevented cell death with parallel decrease in [Ca(2+)](i). Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca(2+)](i) through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA(2), A-SMase, and PKC, or to the generation of ROS.

Contrasting Effects of Phosphatidylinosital-and Phosphatidylcholine-Specific Phospholipase C on Apoptosis in Cultured Endothelial Cells

In the authors' previous studies, they found that phosphatidylcholine-specific phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (PI-PLC) played contrary roles in the apoptosis of vascular endothelial cells (VECs), but the mechanism underlying the phenomenon remains unclear. To address this question, in this study, the authors investigated the changes of cell cycle distribution, the expression of P53, and the phosphorylation of Akt when PI-PLC was inhibited by its specific inhibitor compound 48/80, and they also examined the phosphorylation of Akt when VEC apoptosis was inhibited by D609, a specific inhibitor of PC-PLC. The results showed that suppression of PI-PLC promoted VEC apoptosis by inhibiting Akt phosphorylation, elevating P53 expression, and affecting the cell cycle distribution. Contrarily, suppression of PC-PLC promoted the phosphorylation of Akt. The data suggested that PI-PLC and PC-PLC might control the apoptosis by jointly regulating Akt phosphorylation, P53 expression, and affecting cell cycle in VECs.

PKCα Depletion in RAW264.7 Macrophages Following Microbial/IFNγ Stimulation Is PC-PLC-Mediated

Under chronic inflammatory conditions, monocytes/macrophages often exhibit a desensitized phenotype, which is characterized by attenuated reactive oxygen species (ROS) production in close association with depletion of protein kinase C alpha (PKC alpha). This behavior has been observed in monocytes derived from septic blood although the stimulus responsible for initiating these alterations remained obscure. Using RAW264.7 macrophages, we provide evidence that components of neither gram-negative nor gram-positive bacteria deplete PKC alpha, whereas the T(H)1 cytokine interferon-gamma (IFNgamma) does. As shown by western blot analysis, lipopolysaccharide, as well as lipoteichoic acid, did not alter PKC alpha expression, but IFNgamma dose-dependently decreased PKC alpha protein level. Taking into consideration that diacylglycerol and Ca2+ as established PKC alpha activators are released in response to phospholipase C activation, we pretreated cells with the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl potassium xanthate (D609) and the phosphatidylinositol-specific phospholipase C inhibitor 1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In cells preincubated with D609, IFNgamma-mediated PKC alpha depletion was attenuated, whereas U73122 did not impair this process. Moreover, phorbol 12-myristate 13-acetate-initiated ROS formation, which was attenuated in macrophages pretreated with IFNgamma, was restored in the presence of the PC-PLC inhibitor. These results suggest that IFNgamma causes PC-PLC stimulation, diacylglycerol release, Ca2+ influx, and concomitant PKC alpha activation, which subsequently depletes PKC alpha. Strategies to antagonize IFNgamma might be helpful to prevent monocyte/macrophage desensitization.

Phosphatidylcholine (PC)-specific Phospho-Lipase C (PC-PLC) is required for LPS-mediated macrophage activation

Lipid rafts are sphingolipid-rich membrane microdomains critical to the assembly of the TLR4 receptor complex during LPS exposure within the macrophage. Previously we have demonstrated that activation of the atypical PKC, PKC-ζ, is required for LPS-mediated TLR4 receptor assembly and activation. However, the mechanism in which this kinase is activated following LPS exposure leading to TLR4 receptor assembly and activation remains unknown. The purpose of this study is to determine if the sphingolipid metabolite ceramide produced by PC-PLC is required for activation of this process by LPS. Methods. Differentiated THP-1 cells were stimulated with 100 ng/ml of LPS. Selected cells were pretreated with 100 μM of the PC-PLC inhibitor tricylodecan-9-yl xanthogentate (D609). Lipid raft and cellular protein were extracted and analyzed for TLR4, and PKC-ζ and MAPK activation. TLR4 mobilization to lipid rafts was verified by immunohistochemistry. Harvested supernatants were analyzed by ELISA for TNF-μ production. Results. LPS led to the phosphorylation of PKC-ζ and the mobilization of TLR4 to lipid rafts as demonstrated by immunoblotting and immunohistochemistry. This receptor complex assembly was followed by the activation of the MAPK family, consisting of ERK 1/2, p38, and JNK, and the production of TNF-μ. Pretreatment with D609 resulted in abolished LPS-mediated PKC-ζ phosphorylation, and attenuated LPS-mediated TLR4 lipid raft mobilization (figure), MAPK activation, and TNF-μ production ( P > 0.05). Conclusion. TLR4 receptor assembly occurs following LPS exposure. Assembly of the TLR4 receptor complex requires the generation of ceramide by PC-PLC. Thus, modulation of sphingholipid conversion to ceramide by PC-PLC may serve as a therapeutic target for modulation of macrophage activation during sepsis.

Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts

To investigate expression, subcellular localization and mechanisms of translocation of phosphatidylcholine-specific phospholipase C (PC-PLC) during the cell proliferative response, biochemical, immunoblotting, and immunofluorescence analyses were performed on quiescent and mitogen-stimulated NIH-3T3 fibroblasts. Platelet-derived growth factor (PDGF), insulin and 12- O-tetradecanoylphorbol-13-acetate induced, in 10–60 min, PC-PLC translocation from a perinuclear cytoplasmic area to the plasma membrane. Following cell exposure to PDGF (60 min), the overall PC-PLC expression increased up to 2–3×, while the enzyme activity increased 5× in total cell lysates, 2× in the plasma membrane, and 4× in the nucleus; moreover, confocal laser scanning microscopy showed a progressive externalization of PC-PLC on the outer plasma membrane surface and its accumulation in the nuclear matrix. Pre-incubation of cells with the PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609), before PDGF-stimulation, not only reduced the enzyme activity in total cell lysates as well as in plasma membrane and nuclear fractions, but also blocked the mechanisms of PC-PLC subcellular redistribution. These effects were associated with a D609-induced long-lasting cell cycle block in G o.

Priming of alveolar macrophage respiratory burst by H(2)O(2) is prevented by phosphatidylcholine-specific phospholipase C inhibitor Tricyclodecan-9-yl-xanthate (D609).

The respiratory burst in alveolar macrophages is enhanced in vitro by pre-exposure to nontoxic concentrations of hydroperoxides before stimulation by an agonist, which may represent a feed-forward regulatory mechanism. Tricyclodecan-9-yl-xanthate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), suppresses this priming of the respiratory burst by pre-exposure to H(2)O(2) in NR8383 alveolar macrophages (up to 100 microM D609, 400 nmol of H(2)O(2) added to 5 x 10(6) cells 15 min before stimulation with ADP). D609 has potential as an antioxidant due to its dithiocarbonate functional group that allows it to slowly react with H(2)O(2) and rapidly reduce cytochrome c, which interferes with a common assay for the respiratory burst. Nonetheless, the antioxidant properties of D609 do not account for its inhibition of priming of the respiratory burst by H(2)O(2). Reduction of nitro blue tetrazolium is the basis for an assay for superoxide production with which D609 does not interfere. With this assay, it was found that D609 does not inhibit the respiratory burst per se, but prevents its enhancement by pre-exposure to H(2)O(2). Consistent with a role of diacylglycerol generation by phospholipase C, this enhancement was mimicked by pre-exposure to phorbol ester. In contrast with priming, receptor-mediated stimulation of the respiratory burst depends on the better characterized phosphatidylinositol-specific phospholipase C. Priming of the respiratory burst by H(2)O(2) joins the list of inflammatory responses that are inhibited by D609. Nevertheless, the results herein indicate that caution should be exercised in the interpretation of the effects of D609 to consider both antioxidant effects and inhibition of PC-PLC.

Cellular localization and functional role of phosphatidylcholine-specific phospholipase C in NK cells.

Although several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (M(r) 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity.

Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C.

We previously reported that interleukin-1alpha (IL-1alpha)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1alpha itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1alpha-induced IL-6 synthesis in MC3T3-E1 cells. IL-1alpha time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1alpha-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1alpha. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1alpha-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1alpha-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1alpha was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1alpha. These results strongly suggest that IL-1alpha-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1alpha itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.

Signaling Through CD5 Involves Acidic Sphingomyelinase, Protein Kinase C-ζ, Mitogen-Activated Protein Kinase Kinase, and c-Jun NH2-Terminal Kinase

AbstractThe CD5 lymphocyte surface glycoprotein is a coreceptor involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. The molecular basis for its modulatory properties is not yet well understood. In the present study we describe early biochemical events triggered by CD5 stimulation, which include the phosphatidylcholine-specific phospholipase C (PC-PLC)-dependent activation of acidic sphingomyelinase (A-SMase) in normal and lymphoblastoid T and B cells. The functional coupling of PC-PLC and A-SMase is demonstrated by the abrogation of A-SMase activation by 1) xanthogenate tricyclodecan-9-yl (D609), a selective inhibitor of PC-PLC, and 2) replacement of several C-terminal serine residues (S458, S459, and S461) present in the cytoplasmic tail of CD5 that are known to be critical for PC-PLC activation. Additionally, we demonstrate that activation of protein kinase C-ζ (PKC-ζ) and members of the mitogen-activated protein kinase (MAPK) cascade (MAPK kinase and c-Jun NH2-terminal kinase), but not the NF-κB, are downstream events of the CD5 signaling pathway. A-SMase, PKC-ζ, and MAPK family members are key mediators of cell responses as diverse as proliferation, differentiation, and growth arrest and may contribute to CD5-mediated modulation of TCR or BCR signaling.

A Phosphatidylcholine-Specific Phospholipase C Regulates Activation of p42/44 Mitogen-Activated Protein Kinases in Lipopolysaccharide-Stimulated Human Alveolar Macrophages

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death.

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Function of the p55 tumor necrosis factor receptor "death domain" mediated by phosphatidylcholine-specific phospholipase C.

Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammation that has been implicated in the pathogenesis of devastating clinical syndromes including septic shock. We have investigated the role of a TNF-responsive phosphatidylcholine-specific phospholipase C (PC-PLC) for the cytotoxic and proinflammatory activity of TNF. We show here that the cytotoxicity signaled for by the so-called "death domain" of the p55 TNF receptor is associated with the activation of PC-PLC. The xanthogenate tricyclodecan-9-yl (D609), a specific and selective inhibitor of PC-PLC, blocked the cytotoxic action of TNF on L929 and Wehi164 cells. In vivo, D609 prevented both adhesion molecule expression in the pulmonary vasculature and the accompanying leukocyte infiltration in TNF-treated mice. More strikingly, D609 protects BALB/c mice from lethal shock induced either by TNF, lipopolysaccharide, or staphylococcal enterotoxin B. Together these findings imply PC-PLC as an important mediator of the pathogenic action of TNF, suggesting that PC-PLC may serve as a novel target for anti-inflammatory TNF antagonists.

Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal.

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.

Docosahexaenoic acid selectively attenuates induction of vascular cell adhesion molecule-1 and subsequent monocytic cell adhesion to human endothelial cells stimulated by tumor necrosis factor-alpha.

Incorporation of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) but not eicosapentaenoic acid or n-6 arachidonic acid into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor-alpha (TNF-alpha)-induced surface expression of vascular cell adhesion molecule-1 (VCAM-1). In parallel, DHA inhibited TNF-alpha-stimulated monocytic U937 cell adhesion to HUVECs but did not affect TNF-alpha- or interferon gamma-induced expression of intercellular adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 or VCAM-1 induction by interleukin-1 beta. DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-1 mRNA by TNF-alpha. VCAM-1 induction is regulated by activation of nuclear factor-kappa B, which can be mediated by a TNF-alpha-responsive phosphatidylcholine-specific phospholipase C (PC-PLC). Gel-shift analysis showed inhibition of TNF-alpha-induced nuclear factor-kappa B mobilization by DHA. While the PC-PLC inhibitor D609 dose-dependently prevented VCAM-1 induction by TNF-alpha, 1,2-diacyl-glycerol (DAG) stimulated VCAM-1 expression, suggesting that VCAM-1 induction by TNF-alpha may be mediated by activation of PC-PLC. Treatment with DHA resulted in a fourfold enrichment in PC. In addition, DHA or D609 but not eicosapentaenoic acid or arachidonic acid suppressed activation of PC-PLC by TNF-alpha, estimated as [14C]DAG synthesis in prelabeled HUVECs. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF-alpha and subsequent monocytic cell adhesion by inhibition of TNF-alpha-stimulated PC-PLC activation in HUVECs.