Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.     

Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity

This laboratory first provided evidence for a potential signal transduction pathway involving sphingomyelin and its derivatives (Kolesnick, R.N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). Recently, this laboratory demonstrated the existence of the novel sphingolipid ceramide 1-phosphate in human leukemia (HL-60) cells. Ceramide 1-phosphate was synthesized from ceramide derived from sphingomyelin but not glycosphingolipids. This suggested that a specific pathway extended from sphingomyelin to ceramide 1-phosphate. The present studies provide additional support for this notion by demonstrating the existence of a ceramide kinase activity distinct from diacylglycerol (DG) kinase in HL-60 cells. Microsomal membranes contained a kinase activity that phosphorylated ceramide but not 1,2-DG in the presence of physiologic and higher Ca2+ concentrations (60 nM-3 mM). Kinetic analyses demonstrated an apparent Vmax for ceramide and ATP of 70 pmol.min-1.mg protein-1; apparent Km values were 45 and 25 microM, respectively. The pH optimum was within the physiologic range (pH 6-8). Magnesium but not other divalent cations (Mn2+, Ba2+, Cd2+, Zn2+) also stimulated ceramide phosphorylation. Magnesium also induced 1,2-DG phosphorylation. Since DG kinase is a Mg2(+)-stimulable enzyme that may utilize ceramide as substrate, additional studies separated calcium-dependent ceramide kinase from DG kinase activity. 1,2-DGs competitively inhibited magnesium- but not calcium-dependent ceramide phosphorylation. Hence, calcium-dependent ceramide kinase activity neither utilized DG as substrate nor was inhibited by DG. These activities were physically separable. Both activities were solubilized by n-octyl-beta-D-glucopyranoside and stabilized by glycerol. Ceramide kinase activity bound weakly to a DEAE-cellulose anion exchange column and eluted with 4-fold purification as a single peak of activity in the flow-through and 0.05 M NaCl elutions. In contrast, the majority of DG kinase activity bound more tightly and was recovered as a broad peak in the 0.2-0.35 M NaCl elutions. These studies demonstrate the existence of a ceramide kinase activity in HL-60 cells which is functionally and physically separable from DG kinase. These studies provide further support for the notion of a specific pathway from sphingomyelin to ceramide 1-phosphate.     

Phosphatidic acid that accumulates in platelet-derived growth factor-stimulated Balb/c 3T3 cells is a potential mitogenic signal.

We developed a monoclonal antibody specific to phosphatidic acid (PA). Using this antibody, a novel method to quantify trace amounts of PA was achieved. With the method, PA can be measured in the range of 20-500 pmol. We applied this method to quantify changes in PA levels in Balb/c 3T3 cells stimulated by platelet-derived growth factor. PA contents were very low in quiescent cells and dramatically increased with time up to 15 min. On the other hand, a biphasic diacylglycerol (DG) increase was found. The early phase showed a transient small peak of DG at 30 s followed by a decrease to 1 min. In the second phase, DG accumulated gradually but very markedly up to 15 min. Treatment with propranolol, a PA phosphohydrolase inhibitor, enhanced the accumulation of PA and inhibited the formation of DG in the second phase. However, R59022, a DG kinase inhibitor, did not influence the accumulation of DG or PA, suggesting that platelet-derived growth factor stimulates mainly phospholipase D-catalyzed hydrolysis of phospholipids rather than phospholipase C-catalyzed hydrolysis in the second phase. PA, even after contaminating lyso-PA was removed, could stimulate DNA synthesis, although lyso-PA was 25 times more potent. Moreover, phospholipase D was found to be a much stronger mitogen than phospholipase C. Phospholipase D treatment caused a biphasic accumulation of PA. PA levels reached a maximum at 1 h, and then decreased between 1 and 2 h; finally, there was a gradual elevation up to 10 h. In this case, there was no significant DG accumulation. On the other hand, phospholipase C treatment induced only DG accumulation without any significant change in PA. These results indicate that PA accumulation, rather than an increase in DG, correlates well with mitogenesis.     

A rise and fall in 1,2-diacylglycerol content signal hexamethylene bisacetamide-induced erythropoiesis.

Previous studies have suggested a role for protein kinase C (PKC) during induction of murine erythroleukemia cell (MELC) differentiation by hexamethylene bisacetamide (HMBA) (Melloni, E., Pontremoli, S., Viotti, P. L., Patrone, M., Marks, P. A., and Rifkind, R. A. (1989) J. Biol. Chem. 264, 18414-18418). The present studies assess the effect of HMBA on the content of 1,2-diacylglycerol (DG), the physiologic activator of PKC, in MELC variants. Exposure of parental Sc9 cells to HMBA induced a rapid rise and fall in DG content. The DG level increased within seconds from 225 pmol.10(6) cells-1 to a maximum of 305 pmol.10(6) cells-1 at 5 min. Thereafter, DG content fell reaching control levels at 30 min and 46% of control at 4 h. Similar DG elevations were detected in HMBA-resistant, phorbol ester-resistant, and vincristine-resistant MELC lines. Early DG elevation was followed by the characteristic rapid fall in both the phorbol ester-resistant and vincristine-resistant lines, both of which differentiate rapidly in response to HMBA. In contrast, in an HMBA-resistant MELC the DG level failed to fall for at least 10 h. Selection of HMBA-resistant cells for vincristine resistance restores both HMBA sensitivity and the rapid fall in DG content. Addition of a synthetic DG, 1-oleyl-2-acetyl glycerol (OAG), along with HMBA and every 2 h for the next 48 h blocked differentiation, as measured by accumulation of benzidine-reactive cells or by the commitment assay in methyl-cellulose. However, if addition of OAG was delayed for just a few minutes, until endogenous DG levels began to fall, differentiation was no longer inhibited. Rapid elevation of DG content is the earliest reported event during HMBA action and a subsequent fall in the DG content appears to be a critical step in the process of commitment to terminal differentiation.     

Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D.

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.     

Diacylglycerol mass measurements in stimulated HL-60 phagocytes

The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.     

Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin

Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from spingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate. In sum, these studies demonstrate the novel phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from sphingomyelin to ceramide 1-phosphate via the phosphorylation of ceramide.     

Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide.

We recently demonstrated that sphingosine enhances interleukin-1 beta (IL-1)-mediated prostaglandin E2 (PGE2) production in human dermal fibroblasts (Ballou, L. R., Barker, S. C., Postlethwaite, A. E., and Kang, A. H. (1990) J. Immunol. 145, 4245-4251). Because sphingosine and ceramide are interconvertable, we extended previous studies by treating cells with C2-ceramide (C2-cer), a membrane-soluble analogue of ceramide, and found that C2-cer stimulates IL-1-mediated PGE2 production to the same degree as sphingosine. In an effort to elucidate the mechanistic basis by which sphingosine and C2-cer affect PGE2 production, we examined the effect of these molecules on the expression of genes encoding cyclooxygenase (EC 1.14.99.1, Cox) and phospholipase A2 (EC 3.1.1.4, PLA2), the rate-limiting enzymes in PGE2 biosynthesis. We found that sphingosine and C2-cer treatment resulted in an 8-fold induction of Cox mRNA within 1-2 h which declined thereafter; concomitant changes in Cox protein were also observed. In contrast, expression of phospholipase A2 remained unaltered. We also found that IL-1-mediated PGE2 production was dramatically enhanced in cells treated simultaneously with sphingomyelinase which led us to directly test the effect of IL-1 on sphingomyelin turnover. IL-1 treatment induced the hydrolysis of a significant fraction of prelabeled sphingomyelin which was accompanied by increased levels of intracellular ceramide. Taken together, our results suggest that enhanced Cox expression may account for the observed enhancement of IL-1-mediated PGE2 production by sphingosine and C2-cer. These data also suggest that endogenous sphingomyelin metabolites, generated in response to IL-1, may play an important role in IL-1 signal transduction.     

Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor.

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.     

Activation of the human neutrophil by calcium-mobilizing ligands. II. Correlation of calcium, diacyl glycerol, and phosphatidic acid generation with superoxide anion generation

Calcium and protein kinase C (Ca2+/phospholipid-dependent enzyme) have been proposed to act as signals in triggering superoxide anion (O2-) generation by neutrophils. We have probed the adequacy and necessity of calcium and diacylglycerol (DG), activators of protein kinase C, in eliciting O2- generation and degranulation. Activation of neutrophils by the ligand 10(-7) M fMet-Leu-Phe triggered elevation of cytosolic calcium (fura-2) and a rapid, biphasic increase in labeled DG in [14C]glycerol and [3H]arachidonate prelabeled cells. Buffering of the fMet-Leu-Phe-induced elevation of cytosolic calcium with MAPTAM (a cell permeant EGTA analogue) inhibited O2- generation by 90% and degranulation by 50%, concordant with a role of calcium in signaling. However, buffering the increase in calcium also decreased DG. Since phosphatidylinositol 4,5-bisphosphate breakdown in response to fMet-Leu-Phe was not inhibited and phosphatidic acid levels were enhanced in MAPTAM pretreated cells, the removal of calcium may enhance further DG metabolism. Thus, a requirement for calcium could not be differentiated from a requirement for DG, and the profound inhibition of O2- generation in the presence of MAPTAM may reflect removal of DG. Four stimuli, fMet-Leu-Phe, 10(-7) M leukotriene B4, 100 micrograms/ml concanavalin A, and 200 nM ionomycin elevated cytosolic calcium and triggered release of specific granules, but only fMet-Leu-Phe and concanavalin A triggered substantial O2- generation. Nevertheless, all four stimuli significantly increased labeled DG. Therefore, elevated DG and elevated calcium may be necessary but do not appear adequate to elicit O2- generation. Only fMet-Leu-Phe and concanavalin A triggered generation of phosphatidic acid (PA) together with DG. Correlation of O2- generation with PA may reflect a requirement for PA per se or for a specific pool of DG that can be further metabolized to PA.     

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle.

Synthesis of polyphosphoinositides has been studied in transverse (T-) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [gamma-32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 +/- 0.8 and 71.9 +/- 9.8 pmol PtdInsP/mg per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and pCa 6. PtdInsP2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 +/- 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdInsP2, a deacylated form of PtdInsP2 which occurs in nearly equal quantities in both T-tubule and SR membranes, may result from a mechanism independent of phospholipase A2.     

Elevated cytosolic Ca2+ activates phospholipase D in human platelets

We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.     

Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation.

To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.     

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.     

Diacylglycerol Kinase δ Phosphorylates Phosphatidylcholine-specific Phospholipase C-dependent,Palmitic Acid-containing Diacylglycerol Species in Response to High Glucose Levels

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.     

Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation.