Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation

In addition to its cytotoxic/cytostatic action on many tumor cells in vitro, tumor necrosis factor (TNF) was recently shown to stimulate the growth of some types of cells in culture. We examined the action of TNF in BALB/c 3T3 cells which have been used extensively to study growth regulation. In subconfluent, rapidly dividing 3T3 cultures, murine (Mu) TNF was cytotoxic, while human (Hu) TNF had virtually no antiproliferative action on the cells. In contrast, in density-arrested BALB/c 3T3 cells maintained in a chemically defined, serum-free medium, MuTNF produced a dose-dependent stimulation of DNA synthesis. In stimulating DNA synthesis, MuTNF acted synergistically with both epidermal growth factor or platelet-derived growth factor. While stimulating DNA synthesis in quiescent 3T3 cultures, high doses of MuTNF (100 or 10 ng/ml) were also cytotoxic for a portion of the cells in the same cultures. Cytotoxicity was apparent 2 h after the addition of MuTNF, well before the onset of DNA synthesis. BALB/c 3T3 cell variants selected for their resistance to the cytotoxic action of MuTNF retained the capacity to respond to the mitogenic action of MuTNF, indicating that the stimulation of DNA synthesis by TNF is not a consequence of a TNF "wounding effect." Addition of TNF to density-arrested 3T3 cells resulted in the release of free arachidonic acid and palmitic acid from the cells. Quinacrine, a phospholipase inhibitor, inhibited both cytotoxicity and DNA synthesis in response to TNF, and melittin, a phospholipase activator, mimicked both the cytotoxic and mitogenic actions of TNF in quiescent BALB/c 3T3 cells. These results suggest that phospholipid breakdown is part of the essential early signal transduction events required both for the cytotoxic and mitogenic response to TNF action.     

Modulation of tumor necrosis factor-alpha cytotoxicity in L929 cells by bacterial toxins, hydrocortisone and inhibitors of arachidonic acid metabolism

L929 cells were incubated with tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of various inhibitors of arachidonic acid metabolism. The addition of either hydrocortisone or nordihydroguaiaretic acid (NDGA) decreased the cytotoxic effect of TNF-alpha but exogenously added arachidonate or linoleate, indomethacin and eicosatetraynoic acid (ETYA) were without effect. While it was found that TNF-alpha stimulated arachidonic acid release, no metabolites of this fatty acid could be evidenced. Cytotoxicity of TNF-alpha could also be decreased by the addition of either cholera or pertussis toxin. These results suggest that a GTP-binding protein is involved in the cytotoxic action of TNF-alpha. Arachidonic acid, released possibly by a phospholipase A2, might also play a role, but probably not via its conversion to known metabolites.     

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

Arachidonic Acid Liberated by Diacylglycerol Lipase Is Essential for the Release Mechanism in Chromaffin Cells from Bovine Adrenal Medulla

Chromaffin cells from bovine adrenal medulla secrete catecholamines on stimulation with acetylcholine. In addition to the activation of the phosphatidylinositol cycle, arachidonic acid is generated, which was thought to be the result of phospholipase A2 activation. We have demonstrated in isolated plasma membranes of these cells that arachidonic acid is generated by a two-step reaction of diacylglycerol and monoacylglycerol lipase splitting diacylglycerol, which originates from the action of phospholipase C on phosphatidylinositols. No phospholipase A2 activity could be detected in plasma membranes so far. External addition of arachidonic acid increases the release in the absence and in the presence of agonist. Inhibition of the diacylglycerol lipase by RHC 80267 suppresses the catecholamine release, which is restored on addition of arachidonic acid. This effect, however, is reversed by lipoxygenase inhibitors, indicating that it is not arachidonic acid itself, but one of its lipoxygenase products, that is essential for inducing exocytosis.     

Phospholipase A2 activity is not involved in the tumor necrosis factor-triggered apoptotic DNA fragmentation in bovine aortic endothelial cells.

Using the arachidonic acid release as a probe of phospholipase A2 activity, we tested the involvement of this enzyme in the TNF-triggered apoptotic cell death in the bovine aortic endothelial cells. We observed that TNF induced a liberation of arachidonic acid from these cells which was comparable to that obtained from the L929 cells. An augmentation of the amount of released arachidonic acid or the reduction of the TNF-stimulated phospholipase A2 activity did not modify the TNF-induced DNA fragmentation in the endothelial cells. We suggest that these events are not required for TNF apoptotic cytotoxicity in the endothelial cells.     

Products of the phospholipase A(2) pathway mediate the dihydrorhodamine fluorescence response evoked by endogenous and exogenous peroxynitrite in PC12 cells

A short-term exposure of PC12 cells to tert-butylhydroperoxide promotes a rapid oxidation of dihydrorhodamine sensitive to nitric oxide synthase inhibitors and peroxynitrite scavengers. This response was not directly caused by peroxynitrite, but rather appeared to be mediated by peroxynitrite-dependent activation of phospholipase A(2). The following lines of evidence support this inference: (i) the peroxynitrite-dependent dihydrorhodamine fluorescence response was blunted by low concentrations of two structurally unrelated phospholipase A(2) inhibitors; (ii) under similar conditions, the phospholipase A(2) inhibitors prevented release of arachidonic acid; (iii) low levels of arachidonic acid restored the dihydrorhodamine fluorescence response in nitric oxide synthase- as well as phospholipase A(2)-inhibited cells; (iv) the dihydrorhodamine fluorescence response induced by authentic peroxynitrite was also blunted by phospholipase A(2) inhibitors and restored upon addition of reagent arachidonic acid. We conclude that endogenous, or exogenous, peroxynitrite does not directly oxidize dihydrorhodamine in intact cells. Rather, peroxynitrite appears to act as a signalling molecule promoting release of arachidonic acid, which in turn leads to formation of species causing the dihydrorhodamine fluorescence response.     

Inhibitors of phospholipase A2 block the stimulation of protein synthesis by insulin in L6 myoblasts.

Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

Resistance to killing by tumor necrosis factor in an adipocyte cell line caused by a defect in arachidonic acid biosynthesis

We have found that TA1-R6, which are resistant to the cytotoxic effects of tumor necrosis factor (TNF) in the presence of cycloheximide (Reid, T. R., Torti, F., and Ringold, G. M. (1989) J. Biol. Chem. 264, 4583-4589), have reduced ability to release arachidonic acid (20:4) from membrane phospholipids in response to either TNF or the calcium ionophore A23187 treatment. However, no defect in the activity of phospholipase A2, the principal enzyme responsible for the release of 20:4 from phospholipids, was observed in these cells. Detailed biochemical characterization of these TNF-resistant cells has revealed that these cells are unable to synthesize 20:4 endogenously because of a defect in delta 6-desaturase, the rate-limiting enzyme of 20:4 biosynthesis. This deficiency leads to a marked decrease in the steady-state levels of 20:4 present in choline-containing phospholipid (PC) and ethanolamine-containing phospholipid (PE). The TA1-R6 cells, however, are capable of incorporating exogenous 20:4 into PC and PE, and when loaded in such manner they become significantly more sensitive to the cytotoxic effects of TNF in the presence of cycloheximide. Therefore, the release of arachidonic acid from phospholipids appears to be a critical element in the signaling pathway utilized by TNF and is essential to the rapid cytotoxic response elicited by TNF in the absence of protein synthesis in wild-type TA1 cells.     

Cyclic GMP analogs inhibit gamma thrombin-induced arachidonic acid release in human platelets

Elevation in intracellular cyclic GMP levels is the proposed proximal mechanism for the vasodilatory and platelet inhibitory action of nitrovasodilators and of nitric oxide, the putative endothelium-derived relaxing factor. In this study, the stable cyclic GMP analogs, 8-bromo-cGMP and N2, 2'-O-dibutyryl-cGMP were found to inhibit the release of [3H]-arachidonic acid from gamma thrombin-stimulated human platelets in a time- and dose-dependent manner. Inhibition of the formation of arachidonic acid metabolites, 12-HETE and thromboxane B2, paralleled that of arachidonic acid release and was accompanied by a dose-dependent inhibition of platelet aggregation. The formation of phosphatidic acid, a metabolite of phospholipase C, however, was relatively preserved. At a concentration of 8-bromo-cGMP (2 mM) that produced near-total inhibition of arachidonic acid release, phosphatidic acid formation remained at 60% of control levels. Thus, cGMP analogs have a preferential inhibitory effect on the release and subsequent metabolism of arachidonic acid. The phospholipase A2/arachidonic acid pathway appears to be an important target for the physiologic action of cGMP, and EDRF, and for the pharmacologic action of nitrovasodilators.     

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se.

L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We [Suffys et al. (1987) Biochem. Biophys. Res. Commun. 149, 735-743], as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A2-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, delta 4 Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings. 1. TNF treatment of cells, prelabelled for 24 h with [3H] delta 4Ach or [14C] delta 3Ach (delta 3Ach identical to 5,8,11-icosatrienoic acid) resulted in an early, time-dependent and concentration-dependent release of radioactivity in the supernatant preceding actual cell death. The extent of this response was moderate, albeit reproducible and significant. Analysis of the total lipid fraction from cells plus supernatant revealed that only release of arachidonic acid from phospholipids, but not its metabolization was induced by TNF. However, the release of less unsaturated fatty acids, such as linoleic acid (Lin) or palmitic acid (Pam), was not affected during the first hours after TNF addition. 2. An L929 subclone, selected for resistance to TNF toxicity, was found to be defective in TNF-induced delta 4Ach libration. 3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of delta 4Ach. 4. Release of delta 4Ach was not restricted to TNF; the addition of serum to the cells also induced release of fatty acids into the medium. In this case, however, there was no specificity, as all fatty acids tested, including Lin and Pam, were released. 5. Inhibition of PL-A2 activity by appropriate drugs markedly diminished TNF-induced delta 4Ach release and resulted also in a strong decrease in TNF-induced cytotoxicity. 6. Other drugs, including serine protease inhibitors, which strongly inhibit TNF-induced cytotoxicity, also decreased the TNF-induced delta 4Ach release, whereas LiCl potentiated both TNF-mediated effects. 7. Protection of cells against TNF toxicity by means of various inhibitors was not counteracted by addition of exogenous fatty acids, including delta 4Ach.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid

Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5-lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact human neutrophils but not in the cell-free system

The mechanism involved in the stimulation of NADPH-oxidase by arachidonic acid (AA) in intact human neutrophils was studied and compared with that involved in a cell-free system. [3H]-AA was released from pre-labeled cells upon AA stimulation, and phospholipase A2 inhibitors reduced in parallel the release of [3H]-AA and superoxide. Cyclooxygenase, lipoxygenase or protein kinase inhibitors failed to affect either response. In a cell-free system, no release of [3H]-AA was observed after AA addition, whereas NADPH-oxidase was activated; the generation of superoxide was not inhibited by phospholipase inhibitors and was not initiated by adding phospholipase A2 to the preparation. Thus AA stimulates NADPH-oxidase through a phospholipase A2 mediated pathway in intact cells, but activates the oxidase independent of phospholipase A2 in a broken cell system, suggesting distinctive mechanisms of activation for each system.     

The formation of [3H]inositol phosphates in human platelets by palmitoyl lysophosphatidic acid is blocked by indomethacin

The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.     

Arachidonic acid release in renal proximal tubule cell injuries and death

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-¹⁴C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A₂ activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.