Arachidonic acid, a growth signal in murine P815 mastocytoma cells (original) (raw)
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Arachidonic Acid, a Growth Signal in Murine P815 Mastocytoma Cells1
Evidence is presented that inducing P815 murine mastocytoma cells to grow with serum activates a Ca2-stimulated phospholipase A2 and the rapid release of arachidonic acid by the cells. Slower growth was also maintained by arachidonic acid or its immediate precursors or by diacylglycerols when bovine serum albumin replaced the serum. Together, arachidonic acid and l-oleoyl-2-acetylglycerol stimulated growth at the same rate as 10% serum consistent with a role for both arachidonic acid and protein kinase C in the response to serum. Arresting cell growth with N*,O2 -dibutyryladenosine 3',5'-cyclic phos phate and theophylline inhibited the release of arachidonic acid in re sponse to serum, suggesting that cyclic AMP prevents phospholipase ac tivation as one of its pleiotypic effects on growth.
Journal of Biological Chemistry, 2001
The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A 2 expression and activity indicated that cytosolic phospholipase A 2 did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B 4 and significantly greater amounts of PGD 2 than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 ؎ 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.
Journal of Allergy and Clinical Immunology, 1996
We studied arachidonic acid (AA) metabolism during the maturation of bone marrow-derived cultured mast cells (BMCMCs) into mast cells with phenotypic characteristics, which were more similar to those of connective tissue-type mast cells. BMCMCs were maintained in medium containing 100 ng/ml recombinant rat stem cell factor (SCF) for 1 to 6 weeks. After 3 to 4 weeks in SCF, BMCMCs acquired many phenotypic characteristics of maturation, including enlarged size, numerous electron-dense cytoplasmic granules, and a 50-foM elevation in histamine content. Maintenance in SCF for 6 weeks did not significantly alter the amounts Or species of eicosanoids that were produced by BMCMCs stimulated with calcium ionophOre A23187. However, SCFtreated mast cells released 2.6 + 0.13 times more free AA and accumulated 6.4 +_ 1.0 times higher levels of intracellular free AA than did immature BMCMCs not exposed to SCF. There was no increase in the mobilization of other fatty acids (e.g., linoleic or oleic acid), indicating specificity for AA. Moreover, there were no differences between the 5-1ipoxygenase activities of SCF-treated or untreated cells, as assayed in cell homogenates prepared by nitrogen cavitation. Although the total AA content in SCF-treated cells was significantly elevated, the distribution of AA in phospholipid and neutral lipid classes was not altered by SCF treatment. Total phospholipase (PL)A 2 activity increased 85% +_ 11.5% in SCF-treated cells. In homogenates of immature BMCMCs, 51.0% +_ !3.7% of the PLA 2 activity was inhibited by 0.5 mmol/L dithiothreitol, whereas the same concentration of dithiothreitol caused only a 2.2% _+ 10. 7% reduction in the PLA 2 activity in homogenates of SCF-treated BMCMCs (p <-0.05, n = 4). These findings suggest that SCF treatment induces a dithiothreitol-resistant PLA 2 and that this PLA z may contribute to the mobilization of AA that is not further metabolized to eicosanoids.
Endocrinology, 1999
Prior studies have shown that 24,25-dihydroxyvitamin D 3 2 D 3 ] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A 2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A 2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH) 2 D 3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH) 2 D 3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [ 3 H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH) 2 D 3 inhibited [ 3 H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH) 2 D 3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKC␣; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKC␣ was inhibited, and matrix vesicle PKC was stimulated; these effects were blocked by 24,25-(OH) 2 D 3 . Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE 2 , had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH) 2 D 3 . Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. ␥-Linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH) 2 D 3 action in resting zone chondrocytes. (Endocrinology 140: 2991(Endocrinology 140: -3002, 1999) )
Journal of Biological Chemistry, 1996
Through analysis of the effects of defined serum components and growth supplements, we reveal here that the factors in serum responsible for inducing recovery of Ca 2؉ pools and growth in thapsigargin-arrested DDT 1 MF-2 cells are exactly mimicked by the three essential fatty acids, arachidonic, linoleic, and ␣-linolenic acids. The EC 50 values for arachidonic and linoleic acids on growth induction of thapsigargin-arrested cells were the same, approximately 5 M. Nonessential fatty acids, including myristic, palmitic, stearic, oleic, and arachidic acids, were without any effect. Although not proven to be the active component of serum, levels of arachidonic and linoleic acids in serum were sufficient to explain serum-induced growth recovery. Significantly, arachidonic or linoleic acids induced complete recovery of bradykinin-sensitive Ca 2؉ pools within 6 h of treatment of thapsigarginarrested cells. Protein synthesis inhibitors (cycloheximide or puromycin) completely blocked the appearance of serum-induced or arachidonic acid-induced agonistsensitive pools. The sensitivity and fatty acid specificity of Ca 2؉ pool recovery in thapsigargin-arrested cells were almost identical to that for growth recovery. No pool or growth recovery was observed with 5,8,11,14eicosatetraynoic acid, the nonmetabolizable analogue of arachidonic acid, suggesting that conversion to eicosanoids underlies the pool and growth recovery induced by essential fatty acids. The results provide not only further information on the link between Ca 2؉ pools and cell growth but also evidence for a potentially important signaling pathway involved in inducing transition from a stationary to a proliferative growth state.
Biochemistry and Cell Biology, 1997
Our previous studies demonstrated that inhibitors of arachidonate-phospholipid remodeling [i.e. the enzyme CoAindependent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate-phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.
2004
Phospholipase A2 regulation of arachidonic acid levels Availability of free arachidonic acid (AA) is widely recognized as a rate-limiting step in the formation of prostaglandins. This fatty acid is an intermediate of a reacylationldeacylation cycle of membrane phospholipids, the so-called Lands pathway, in which the fatty acid is cleaved from phospholipid by phospholipase Azs (PLA z) and reincorporated by acyltransferases. Whereas in resting cells reacylation dominates, in stimulated cells the dominant reaction is the PLArmediated deacylation. Nevertheless, increased AA reacylation during cellular activation is still very significant, as manifested by the fact that only a minor portion of the free AA released by PLA z is converted into eicosanoids, the remainder being effectively incorporated back into phospholipids. Phagocytic cells generally contain multiple PLA 2 s [1, 2]. Thus the challenge in recent years has been both to identify these PLAzs and to clarify their roles in AA metabolism. A general mechanism for PLAz-regulated AA metabolism in resting and activated cells has emerged from the studies by several laboratories [3, 4], and involves participation of all three major classes of PLA z , namely cPLA z (cytosolic PLA z), iPLA z (Ca 2 + independent PLA z) and sPLA z (secreted PLA z) (Fig. 1). In resting conditions, iPLA 2 accounts for most of the PLA z activity of cells. iPLA z is therefore the dominant PLA z involved in the liberation of fatty acids, including AA, during the continuous recycling of membrane phospholipids that takes place under these conditions. Since, as indicated above, the rate of AA release by iPLA z is lesser than the rate of its reacylation back into phospholipids, no net accumulation of free fatty acid occurs. Stimulation of the cells by receptor agonists results in the activation of cPLA z , which then becomes the dominant PLA 2 involved in AA release. Under these conditions, the rate of AA release clearly exceeds that of reincorporation into phospholipids; hence net accumulation of AA occurs that is followed by its conversion into different oxygenated compounds, collectively called the eicosanoids.
Journal of cellular physiology, 2003
In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA-induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Tw...