Differential effects of platelet-derived growth factor, serum and bombesin on phospholipase D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts.
ABSTRACT: In previous studies, activators of protein kinase C, sphingosine, ATP and various oncogenes were each found to enhance phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here I examined possible stimulation of PtdEtn hydrolysis by various growth-stimulatory agents, including serum, bombesin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. Treatment of NIH 3T3 fibroblasts, prelabelled with [14C]Etn or [32P]PtdEtn, with PDGF-BB resulted in enhanced formation of [14C]Etn or [32P]phosphatidic acid from the respective labelled cellular pools of PtdEtn. A maximal effect (approximately 3-fold stimulation) on PtdEtn hydrolysis was obtained with 50 ng of PDGF/ml after 5 min of treatment. Phosphatidylcholine (PtdCho) was also hydrolysed, although less extensively than PtdEtn, in PDGF-stimulated cells. PDGF-stimulate hydrolysis of both PtdEtn and PtdCho was prevented by prolonged (30 h) treatment of cells with 400 nM-phorbol 12-myristate 13-acetate (PMA). Similar to PDGF, fetal calf serum (1-10%) also stimulated PtdEtn hydrolysis. However, in contrast to PDGF, the effect of serum on PtdEtn hydrolysis (i) was not diminished by pretreatment with PMA, and (ii) was synergistic with that of PMA after a 1 h incubation. Compared with PDGF and serum, bombesin had less effect on PtdEtn hydrolysis, while FGF and insulin had no effects at all. In contrast to PDGF or serum, bombesin inhibited the effect of PMA on PtdEtn hydrolysis.
Project description:The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.
Project description:The tumour-promotion-sensitive (P+) and -resistant (P-) variants of mouse JB6 epidermis-derived cells have often been used to study the requirements for the tumour-promoting effect of PMA. As part of an effort to identify the defect(s) in JB6 P- cells that might prevent the promoting effect of PMA, stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PMA as well as the rate of phospholipid synthesis were compared in three P+ variants, two P- variants and a transformed variant of the JB6 cell line. PMA (5-100 nM) had significantly less stimulatory effect on PtdCho hydrolysis in P- cells than in P+ or transformed JB6 cells. The effects of PMA on PtdEtn hydrolysis in the P+ and P- cell lines were similar, whereas in transformed cells PMA had slightly less effect. Each JB6 cell line was found to contain similar amounts of PtdCho. In contrast, P- cells contained significantly less PtdEtn and a correspondingly higher level of ethanolamine phosphate compared with P+ and transformed cells. P- cells also secreted ethanolamine phosphate into the medium; this process was greatly enhanced by PMA. In the two P- variants the synthesis of PtdEtn from [14C]ethanolamine was reduced to various extents, whereas the rate of PtdCho synthesis was comparable in each JB6 cell line. The synthesis of PtdCho, but not PtdEtn, was greatly stimulated by PMA in both the P+ and P- clones. The results indicate that decreased synthesis/level of PtdEtn and suboptimal functioning of a PtdCho-specific PLD are common characteristics of the P- JB6 cells examined so far. The observed alterations in phospholipid metabolism may play a role in the resistance of P- cells to the tumour-promoting action of PMA.
Project description:Previously, the protein kinase C (PKC) inhibitor sphingosine was found to stimulate phospholipase D (PLD)-mediated hydrolysis of both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts [Kiss & Anderson (1990) J. Biol. Chem. 265, 7345-7350]. Here we examined the possible relationship between the opposite effects of sphingosine on PKC-mediated protein phosphorylation and PLD activation. After treatments for 3-5 min, sphingosine (25 microM) and the PKC activators phorbol 12-myristate 13-acetate (PMA) (100 nM), bryostatin (100 nM) or platelet-derived growth factor (50 ng/ml) synergistically stimulated the hydrolysis of both PtdEtn and PtdCho in NIH 3T3 fibroblasts prelabelled with [14C]ethanolamine or [14C]choline. Inhibition of PMA-induced phospholipid hydrolysis could also be elicited by sphingosine, but this process required prolonged (60 min) treatments of fibroblasts with 40-60 microM-sphingosine. Similarly to sphingosine, the protein phosphatase inhibitor okadaic acid also had either potentiating or inhibitory effects on PMA-stimulated PLD activity, depending on the length of incubation time and the concentration of PMA. Consistent with the presence of an inhibitory component in the overall action of PKC, the PKC inhibitor staurosporine and down-regulation of PKC activity by prolonged (24 h) treatment with PMA similarly enhanced PLD activity. Data suggest that (a) sphingosine may enhance PMA-mediated phospholipid hydrolysis by neutralizing the action of an inhibitory PKC isoform, and that (b) the stimulatory PKC isoform is less sensitive to the inhibitory action of sphingosine.
Project description:The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.
Project description:Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation; this is a major phenomenon since together these metabolites represent 60% of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 microM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 microM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 microM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not into Etn. Increasing exogenous serine to 100 microM decreased the incorporation of radioactive Etn into PtdEtn by only 30%, and the PtdCho level was not affected. 2-Hydroxyethylhydrazine significantly decreased serine incorporation into P-Etn and PtdEtn, whereas Etn was accumulated. No concomitant inhibition of PtdSer or PtdCho labelling from serine occurred, even when PtdEtn formation was decreased by 95%. This indicates that the PtdEtn pool derived from direct serine decarboxylation differed from that derived from PtdSer decarboxylation, and the latter appeared to be preferentially used for PtdCho biosynthesis. Hydroxylamine also inhibited phosphorylation of serine-derived Etn but not that of exogenous Etn. The rate of PtdSer synthesis from 10 microM L-serine was 3.1+/-0.5 and 2.95+/-1.3 nmol/5 h per 10(10) infected cells, whereas L-serine decarboxylation accounted for 7.1+/-1.5 and 9.9+/-3 nmol/5 h per 10(10) infected cells for P. falciparum and P. knowlesi respectively (means+/-S.E.M.). The serine decarboxylating reaction was not detected in other higher eukaryotic cells such as mouse fibroblasts and human lymphocytes. Finally, these results also indicate compartmentalization of phospholipid metabolism in Plasmodium-infected erythrocytes.
Project description:The aim of this study was to clarify the relationship between the stimulatory effects of protein kinase C activators, including phorbol 12-myristate 13-acetate (PMA) and bryostatin, on the hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) and on PtdCho synthesis. The cell lines used were selected because of their differential responses to protein kinase C activators and included rat-1 fibroblasts, untransformed and A-raf-transformed NIH 3T3 fibroblasts and human HL60 leukaemia cells. Exposure of rat-1 and NIH 3T3 fibroblasts to 100 nM-PMA stimulated phospholipase D-mediated hydrolysis of phospholipids about 2- and 6-fold respectively. In contrast, 100 nM-PMA had similar (2.5-3.0-fold) stimulatory effects on PtdCho synthesis in these cell lines. In the untransformed NIH 3T3 cells, both PMA and bryostatin stimulated both phospholipid hydrolysis and PtdCho synthesis, with 100 nM-bryostatin being somewhat less potent than 100 nM-TPA. In contrast, in A-raf-transformed NIH 3T3 cells or in HL60 cells, only TPA, but not bryostatin, stimulated PtdCho synthesis. In these transformed cells, bryostatin had 3-fold, or higher, stimulatory effects on phospholipid hydrolysis. Addition of ionomycin, a Ca2(+)-elevating agent, partially restored the stimulatory effect of bryostatin on PtdCho synthesis, but it failed to modify the effect of bryostatin on phospholipid hydrolysis. These data indicate that increased phospholipid hydrolysis is not necessarily associated with increased PtdCho synthesis.
Project description:Endogenous content of and incorporation of labelled glycerol into alkenylacyl-, alkylacyl- and diacyl-glycerol, -glycerol-3-phosphocholine and -glycero-3-phosphoethanolamine of pulmonary type II cells were measured. On prolonged incubation of type II cells with labelled glycerol, the proportion of label incorporated into the diacyl subclass of these glycerolipids increased and the proportion of label incorporated into the ether lipids declined. Endogenous phosphatidylcholine (PtdCho) of type II cells contained 38.4% of the dipalmitoyl species, but endogenous phosphatidylethanolamine (PtdEtn) only 2.5%. In contrast, similar proportions of labelled glycerol were incorporated into dipalmitoyl-PtdCho and -PtdEtn after short-time incubation but, with prolonged incubation time the proportion of labelled dipalmitoyl-PtdCho increased from 11.3 to 18.8%, whereas that of dipalmitoyl-PtdEtn did not change significantly. Type II cell membranes were found to exhibit cofactor-independent and CoA-mediated transacylations of [1-14C]palmitoyl-lyso-PtdCho and -lyso-PtdEtn. The distribution of label among the palmitic acid-containing species of PtdCho and PtdEtn formed by both transacylation activities was determined. Cofactor-independent and CoA-mediated transacylation showed a strong selectivity for palmitate and arachidonate and a strong discrimination against oleate. The amount (nmol) of dipalmitoyl-PtdEtn formed by both transacylation activities after short-time incubation (2 min) decreased with prolonged incubation time (60 min). In contrast, the nmol of dipalmitoyl-PtdCho formed by cofactor-independent transacylation remains nearly the same after short-time and longer incubation. The nmol of dipalmitoyl-PtdCho formed by CoA-mediated transacylation increased strongly in the same time interval. Beside synthesis de novo via the CDP-choline pathway and reacylation of lyso-PtdCho with palmitoyl-CoA, the CoA-mediated transacylation of lyso-PtdCho may be an effective pathway for the formation of dipalmitoyl-PtdCho in pulmonary type II cells.
Project description:In this work, we determined the effects of sphingosine 1-phosphate (S1P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [14C]ethanolamine-labelled cells, S1P (0.5-5 microM) stimulated PLD-mediated hydrolysis of PtdEtn 1.5-2.1-fold. Down-regulation of protein kinase C by chronic (24 h) treatment of cells with 300 nM PMA, or pretreatments (10 min) with the cell-permeant calcium chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N, N',N'-tetra-acetic acid tetra-acetoxymethyl ester led to the inhibition of S1P-induced PtdEtn hydrolysis. S1P alone was a weak inducer of DNA synthesis, but its effects were enhanced by phosphocholine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 microM) did not modify the mitogenic effect of S1P alone, whereas at 50-100 microM concentrations it actually enhanced the mitogenic effect of PCho via a mitogen-activated protein (MAP) kinase-independent mechanism. In contrast, 5-20 microM concentrations of ethanolamine, which correspond to normal blood ethanolamine levels in humans, strongly inhibited DNA synthesis induced by S1P plus PCho via a MAP kinase-dependent mechanism; importantly, less or no inhibition was observed with 50-100 microM concentrations of ethanolamine. At 5-50 microM concentrations, ethanolamine also inhibited the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1P plus PMA or S1P plus ATP. The results indicate that S1P stimulates PLD-mediated hydrolysis of PtdEtn by a mechanism that may involve a regulatory protein kinase C isoform. Increased formation of ethanolamine by PLD-mediated PtdEtn hydrolysis or by other means may be required for maximal stimulation of DNA synthesis by S1P in the presence of insulin, and particularly PCho.
Project description:We examined the role of protein kinase C alpha (PKC alpha ) in the stimulation of DNA synthesis of Swiss 3T3 cells induced by bombesin, platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA). We found that cells in which this kinase had been down-regulated showed a partially abrogated mitogenic response to bombesin. The response to PDGF was unaltered; however, the response to PMA was completely suppressed. The mitogenic effect of maximal doses of bombesin and PMA combined was greater than that of either agent alone, suggesting that bombesin does not fully activate the PKC pathway. Accordingly, bombesin-induced PKC alpha translocation from cytosol to membranes was partial, while that observed with PMA was essentially complete. Moreover, exposure to Ro-31-8220, a PKC inhibitor, had significantly greater effects on the response to PMA than on that to bombesin. Our findings point out different roles that PKC alpha may play in diversely activated cells: while, in the case of PMA, stimulation of this kinase may be necessary and sufficient to induce proliferation, it appears to be necessary only for a full response to bombesin, and redundant among the mechanisms triggered by PDGF.
Project description:Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; 'TPA'), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5'-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.