Phosphoinositide-specific phospholipase C of a cloned mast cell line, mastocytoma P-815: purification and some characterization

1. Multiple forms of phosphoinositide-phospholipase C (PLC) were isolated from mastocytoma; two cytosolic forms (cPLC-I, Mr 150,000; cPLC-II, Mr 110,000) and two membrane-associated forms (mPLC-I, Mr 85,000; mPLC-II, Mr 85,000). 2. Four PLC forms differently behaved in substrate specificity and effect of GTP-binding proteins.     

Enhancement of 5-lipoxygenase activity in mastocytoma P-815 cells by n-butyrate treatment

Cloned mastocytoma P-815, 2-E-6 cells were used to investigate regulation of 5-lipoxygenase activity. 2-E-6 cells had high 5-lipoxygenase activity with slight 12-lipoxygenase activity. The 5-lipoxygenase activity was increased over 5-fold by treatment of the cells with 1 mM n-butyrate for 18 h. the most effective dose range being 0.1-5.0 mM. Treatment with n-butyrate for 18 h was more effective than treatment for 40 h. Addition of n-butyrate to an untreated cell homogenate had no stimulatory effect. The enhancement of 5-lipoxygenase activity by n-butyrate was accompanied by new synthesis of protein(s). 12-Lipoxygenase activity was not increased so much as 5-lipoxygenase activity by the treatment. This is the first report of stimulation of 5-lipoxygenase activity in cultured cells. The different responses of the two lipoxygenases to n-butyrate treatment strongly suggest that 5-lipoxygenase is a different enzyme from 12-lipoxygenase.     

cDNA-derived amino acid sequence of L-histidine decarboxylase from mouse mastocytoma P-815 cells

The primary structure of L-histidine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-815 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74017, which is larger by about 21,000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.     

Induction of histidine decarboxylase by dexamethasone in mastocytoma P-815 cells

Dexamethasone at a concentration as low as 10 nM significantly increased both the histamine content and histidine decarboxylase activity of cultured mastocytoma P-815 cells. Both effects were clearly seen using several glucocorticoids, which were as effective as dexamethasone. In contrast to that of histamine, the serotonin level of mastocytoma P-815 cells was decreased by treatment with dexamethasone. The dexamethasone-induced increases in histamine content and histidine decarboxylase activity were completely suppressed by the addition of cycloheximide and actinomycin D. Mastocytoma P-815 cells were found to possess binding sites for [3H]dexamethasone in the cytosol (Kd = 15.7 nM) and the nuclei (Kd = 1.26 nM). These results show that glucocorticoids significantly stimulate de novo synthesis of histidine decarboxylase.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

Arachidonic acid mobilization among phospholipids in murine mastocytoma P-815 cells: role of ether-linked phospholipids

The ethanolamine-containing glycerophospholipids, choline-containing glycerophospholipids, and phosphatidylinositol fractions are major sources of arachidonic acid in murine mastocytoma P-815 cloned cells. The choline-linked fraction contained high arachidonic acid contents in 1-O-alkyl-2-acyl- (18%) and 1,2-diacyl-sn-glycero-3-phosphocholine (11%), with smaller amounts in 1-O-alk-1'-enyl-2-acyl species, whereas the arachidonic acid content of the ethanolamine-linked fraction was high in 1-O-alk-1'-enyl-2-acyl (26%) and 1,2-diacyl species (15%) and low in 1-O-alkyl-2-acyl species. The uptake and transfer of [3H]arachidonic acid into the 1,2-diacyl and ether classes of choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids in mastocytoma cells were examined. There was very rapid incorporation of radioactive arachidonic acid into mastocytoma cells that leveled off after 30 min. By labeling cells with [3H]arachidonic acid for 7.5 min, the radioactivity was recovered in the choline-containing glycerophospholipids (43%), phosphatidylinositol (32%), and ethanolamine-containing glycerophospholipids (20%) with little in other phospholipids, neutral lipid, or free fatty acid fractions. Upon reincubation of the mastocytoma cells in the radiolabel-free medium, the [3H]arachidonate radioactivity was gradually lost from the choline-containing glycerophospholipids fraction and, concomitantly, increased in ethanolamine-containing glycerophospholipids. At the zero time of reincubation, most of the radioactivity was recovered in the 1,2-diacyl species of both choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.     

Effect of tunicamycin on functions of PGE1 receptors from mouse mastocytoma P-815 cells

Prostaglandin E1 (PGE1) receptors from mouse mastocytoma P-815 cells were found to bind to a wheat germ agglutinin (WGA)-Agarose column, suggesting that the receptors are glycoproteins. To further elucidate the role of carbohydrate moieties in the PGE1 receptors for their binding activity to ligand, the P-815 cells were treated with tunicamycin, swainsonine or monensin. Tunicamycin, an inhibitor of N-glycosylation, dose- and time-dependently inhibited the binding of PGE1 to mastocytoma P-815 cells. Neither swainsonine, an inhibitor of Golgi mannosidase II, nor monensin, an inhibitor of processing beyond the high mannose stage, altered PGE1 binding properties of the cells. The inhibition of PGE1 binding by tunicamycin was observed when incorporation of [3H]glucosamine into macromolecules was inhibited. The inhibitory effect was not on their affinity but on their number of binding sites. Subcellular distributions of [3H]PGE1-binding activity showed that decreases in the binding activity by tunicamycin were highest in plasma membrane fractions. Treatment of membranes with various endo- and exoglycosidases did not affect PGE1 binding. PGE1-stimulated cyclic AMP accumulation in the cells was also inhibited by tunicamycin. These results suggest that PGE1 receptors of mastocytoma P-815 cells are glycoproteins and that inhibition of N-glycosylation of PGE1 receptors by tunicamycin results in the arrest of the translocation of newly synthesized receptors to the surface of mastocytoma P-815 cells.     

Activation of histidine decarboxylase through post-translational cleavage by caspase-9 in a mouse mastocytoma P-815

L-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp(517)-Asp(518), Asp(550)-Asp(551)) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn(2+) chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.     

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes,EP3 and EP4

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.     

Effect of 7-fluoro prostacyclin,a stable prostacyclin analogue,on cAMP accumulation and prostaglandin binding in mastocytoma P-815 cells

The effect of 7-fluoro proscyclilin (PGI₂-F), a chemically stable analogue of prostacyclin, on cAMP accumulation in and [³H]PGE binding to mastocytoma P-815 cells was compared with those of the Na salt and methyl ester of prostacyclin (PGI₂Na or PGI₂Me), which are rapidly inactivated in aqueous solution or metabolized in the tissue.PGIF was as effective as PGI₂Me, and slightly less effective than PGI₂Na in stimulating cAMP accumulation in mastocytoma cells and rabbit platelets. PGI₂F was also more stable than PGI₂Me or PGI₂Na, and retained its original cAMP elevating activity even after incubation with or without cells for 4 h at 37°C. Cells which had been exposed to PGI₂F and then washed free of unbound reagent continued to produced cAMP for more than 3 h. PGI₂F was also as effective as PGE₁ or PGE₂ in displacing [³H]PGE₂ bound to the cells. Non-competitive inhibition by PGI₂F or PGI₂Me of [³H]PGE₂ binding to the cells, with apparent K_is of 1.29 μM and 1.13 μM, respectively, indicates the presence of different receptors for PGE₂ and for PGI₂F or PGI₂Me in mastocytoma P-815 cells.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Cytosol promotes the guanine nucleotide-induced release of the alpha subunit of Gi2 from the membrane of mouse mastocytoma P-815 cells

Translocation of the alpha subunit of Gi2 from the membrane to the cytosol was studied in mouse mastocytoma P-815 cells. To monitor Gi2 alpha the membrane (300,000 x g pellet) was [32P]ADP-ribosylated with pertussis toxin. Incubation of the [32P]ADP-ribosylated membrane with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a small release (10%) of [32P]ADP-ribosylated Gi2 alpha from the membrane. Whereas cytosol (300,000 x g supernatant) alone had no ability to release the [32P]ADP-ribosylated Gi2 alpha from the membrane, it markedly augmented the release induced by GTP gamma S, about 50% of the total [32P]ADP-ribosylated Gi2 alpha being released by 30 min. The GTP gamma S-induced release and its enhancement by the cytosol were specific for GTP and GTP gamma S. When the cytosol was boiled this promoting activity was lost. The [32P]ADP-ribosylated Gi2 alpha released by the cytosol plus GTP gamma S from the membrane was eluted as a single peak corresponding to a molecular weight of about 100,000 from an Ultrogel AcA 44 column. In contrast, the [32P]ADP-ribosylated Gi2 alpha released by GTP gamma S alone was eluted at the position of Mr = 40,000, but it was eluted at the position of Mr = about 100,000 when it was incubated with the cytosol. Furthermore, Gi2 alpha purified from bovine lung also behaved in a similar way on gel filtration. The addition of thrombin, a stimulant of histamine secretion from mast cells, to mastocytoma cells drastically induced the translocation of Gi2 alpha from the membrane to the cytosol in a pertussis toxin-sensitive manner. These results taken together demonstrate that the cytosol contains some factor(s) that promotes the release of GTP-activated Gi2 alpha from the membrane and that the released Gi2 alpha exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.     

Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells.

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.     

Antibody-mediated enhancement of infection by dengue virus of the P815 murine mastocytoma cell line

Dengue type 2 virus (DEN 2) could replicate only to a limited extent in a murine mastocytoma cell line, P815. The viral multiplication was enhanced 10- to 100-fold by mouse anti-DEN 2 antiserum or anti-DEN 2 type-specific monoclonal antibody diluted beyond their neutralizing titers. Cells incubated with virus-antibody mixtures changed morphologically, developing a mature mast cell-like appearance, 4-5 days after infection. The indirect fluorescent antibody technique showed that the enhancement of infection was caused by an increase in the number of DEN 2-infected cells. This is the first report that cells of mast cell lineage support dengue virus multiplication, and that virus production is enhanced in the presence of anti-dengue antibodies.