Brefeldin A Inhibits Antigen- or Calcium Ionophore-Mediated but not PMA-Induced Phospholipase D Activation in Rat Basophilic Leukemia (RBL-21-13) Cells

Immunobiol., vol. 195, pp. 231-242 (1996)

©

1996 by Gustav Fischer Verlag, Stuttgart

Department of Otolaryngology and Department of Biochemistry, Gifu University School of Medicine, Gifu, Japan

Brefeldin A Inhibits Antigen- or Calcium lonophoreMediated but not PMA-Induced Phospholipase D Activation in Rat Basophilic Leukemia (RBL-2H3) Cells YOSHIKATSU NAKAMURA, SHIGERU NAKASHIMA, TAKAHIKO KUMADA, KATSUHIRO OJIO, HIDEO MIYAT A,

and Y OSHINORI N

OZAWA

Received May 31, 1995 . Accepted in revised form October 18, 1995

Abstract Recent reports have indicated that ADP-ribosylation factor (ARF) plays a role in the regulation of phospholipase D (PLD) activity in the in vitro assay system. Since a fungal metabolite brefeldin A (BFA) is known to interfere with ARF function, the effect of BFA on antigen-induced PLD activation was examined in rat basophilic leukemia (RBL-2H3) cells. BFA inhibited the antigen-induced formation of phosphatidylbutanol (PBut), a specific and stable metabolite produced by PLD activity in a concentration-dependent manner. The maximal inhibition obtained at 10 ftg/ml of the drug was nearly 70 % and further inhibition was not observed at higher concentrations. Ca2 +-ionophore A23187mediated PLD activation was also prevented by BFA. In contrast, BFA failed to inhibit PLD activation in response to 4(3-phorboI12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). This indicates that there are BFA-sensitive and BFA-insensitive pathways leading to PLD activation in RBL-2H3 cells and also that the PKC-mediated pathway may be insensitive to BFA treatment, suggesting the existence of PLD isozymeso BFA inhibited Ag-induced serotonin release at a concentration 20-fold lower than that needed for the inhibition of PLD. Moreover, PMA caused a marked production of PBut, but it failed to elicit secretory response. This implies that PLD may be not a crucial element for secretory responses.

Abbreviations: Ag=antigen; ARF=ADP-ribosylation factor; BFA=brefeldin A; 1,2DC = 1,2-diacylglycerol; Fa:RI = high affinity IgE receptor; CTPyS = guanosine 5'-0(3-thiotriphosphate); 5-HT = 5-hydroxytryptamine; PA = phosphatidic acid; PBut =phosphatidylbutanol; PKC=protein kinase C; PLD=phospholipase D; PMA=4(3phorbol 12-myristate 13-acetate; PTK = protein tyrosine kinase; RBL = rat basophilic leukemia.

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Introduction Mast cells and basophils express high affinity IgE receptor (FcERI). Upon antigen (A g) stimulation, cross-linking of receptor-IgE complexes induces release of chemical mediators such as histamine, serotonin, leukotrienes and prostaglandins. Rat basophilic leukemia (RBL-2H3) cells share many of the properties of mucosal mast cells and are widely used as a model for the study of FCERI-mediated signaling events. Aggregation of FCERI leads to activation of various signal transducing phospholipases (1-3). Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2 ) yielding two second messengers; inositol 1,4,5-trisphosphate (IP) and 1,2-diacylglycerol (DG) (4, 5). In addition, phospholipase D (PLD) is also known to play an important role in the generation of second messengers, phosphatidic acid (P A) and DG in RBL2H3 cells (6,7) and peritoneal mast cells (8). Hydrolysis of phospholipid(s), mainly phosphatidylcholine, by PLD yields PA and choline. PAis further converted to DG by PA phosphohydrolase. PLD is activated in response to either antigen, phorbol ester or calcium ionophore in RBL-2H3 cells (7, 9). We have previously demonstrated that protein tyrosine kinase (PTK) plays a role in PLD activation in RBL-2H3 cells (10). However, its precise regulatory mechanism is not well understood. Recent reports (11-13) have indicated the possible involvement of ADPribosylation factor (ARF), a small GTP-binding protein in the regulation of PLO activity in rat brain and HL60 cells. ARF proteins constitute a subgroup in the Ras superfamily of monomeric GTP-binding proteins (14) and have been implicated as regulators of vesicle-mediated protein traffic (14, 15). Several lines of evidence have suggested the implication of PLD in membrane transport and exocytotic response. A fungal product, brefeldin A (BFA) is known to inhibit vesicular transport from endoplasmic reticulum and has often been used for investigating membrane traffic (16). BFA has recently been shown to inhibit the GDP-GTP exchange on the ARF catalyzed by Golgi membrane proteins (17, 18). The substance, which specifically inhibits ARF activation, may have pharmacological potential for preventing ARF-mediated cellular events. In the present study, we have examined the effects of BFA on PLD activation and serotonin release in RBL-2H3 cells.

Materials and Methods Materials Monoclonal mouse anti-dinitrophenyl (DNP) IgE was obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Ascaris suum antigen coupled with 2,4-dinitrophenyl (DNP-As) was synthesized as described previously (2). Eagle's minimum essential medium (MEM), penicillin and streptomycin were obtained from Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Irvine Scientific (Santa Ana, CA,

BFA inhibits Ag-induced PLD activity in RBL cells . 233 USA). [14C]5-Hydroxytryptamine binoxalate (serotonin, 5-HT) (50 mCi/mmol) and Aquasol-2 were purchased from DuPont/NEN (Boston, MA, USA). [9,10-3H]Palmitic acid (52.41 Ci/mmol) was from Amersham International (Aylesbury, UK). BFA and 413phorbol12-myristate 13-acetate (PMA) was from Sigma (St. Louis, MO, USA). A23187 was from Calbiochem (San Diego, CA, USA). Silica gel 6D and silica gel 60 plates were obtained from Whatman (Clifton, NJ, USA) and Merck (Darmstadt, Germany), respectively. Other reagents were of analytical grade. Cell culture Monolayer cultures of RBL-2H3 cells were maintained in MEM supplemented with 10 % heat-inactivated FBS, penicillin (100 units/ml) and streptomycin (100 [tg/ml) in a humidified atmosphere of 95 % air/5 % COz at 37°C (1, 3). Serotonin release RBL-2H3 cells were incubated with [14C]5-HT (10 nCi/1.5 x 105 cells) for 16h at 37°C. The cultures were washed twice and equilibrated in Tyrode-Hepes solution (NaCI 134 mM, NaHC0 3 12 mM, KC12.9 mM, MgClz 1 mM, CaClz 1.8 mM, NaH zP0 4 0.36 mM, glucose 5.6 mM, Hepes 10 mM, 0.1 % BSA, pH 7.40) for 5 min at 37°C prior to stimulation. After preincubation for 20 min with various concentrations of BFA, the cells were stimulated with Ag (0.1 [tg/ml). Incubation was stopped promptly by removing the medium, followed by addition of 0.2 ml of ice-cold PBS containing 1 % TritonX-100. The radioactivity in the medium and the cells (TritonX-I00 extract) was determined by a liquid scintillation counter (Beckmann LS 6500, Fullerton, CA, USA) with 5 ml of Aquasol-2. The percentage of serotonin release was expressed as the radioactivity in the medium to the sum of that in the medium and the cells. Assay of PLD activity RBL-2H3 cells seeded into 35-mm dishes at 1.5xl06 cells in 1 ml culture medium were labeled with eH]palmitic acid (1 [tCi/dish) for 20 h. For sensitization, anti-DNP IgE (0.3 [tg/ml) was incubated for the last 4 h of radiolabeling. After removal of the radiolabeling medium, the dishes were rinsed three times and equilibrated in Tyrode-Hepes solution for 5 min at 37°C prior to stimulation. Incubations were stopped by removing medium followed by the immediate addition of 1 ml of ice-cold PBS/methanol (2:5, v/v) mixture to the culture dishes. Cells were scraped from the dishes with an additional 1 ml of PBS/ methanol mixture and transferred to a tube containing 700 [tl of chloroform. In addition to its hydrolytic reaction, PLD also catalyzes, in the presence of primary alcohol, a unique and specific transphosphatidylation reaction to generate the corresponding phosphatidylalcohol (19). PLD activity in response to antigen was determined by measuring the formation of eH]phosphatidylbutanol (PBut) in the presence of butanol (0.3 %, v/v) (10). The lipid extraction was performed according to the method of BLIGH and DYER (20). eH]PBut was separated by silica gel 6D plates in a solvent system of upper phase of ethyl acetate12,2,4-trimethylpentane/acetic acid/water (13 : 2: 3: 10, v/v (21). The area corresponding to eH]PBut identified by co-migration with PBut standard was scraped off the plates and the radioactivity was determined by a liquid scintillation counter (Beckmann LS 6500). Measurement of sphingomyelin hydrolysis RBL-2H3 cells seeded into 35-mm dishes at 1.5 x 106 cells in 1 ml culture medium were labeled with eH]palmitic acid (3 f-\Ci/ dish) for 20 h. Cell stimulation and lipid extraction were performed as described above. The extraction lipids were dissolved in 1 ml of

234 . Y. NAKAMURA et al. chloroform/methanol (1:1) and incubated with 1 ml of 0.5 M methanolic NaOH at 3rC for 1 h in order to eliminate ester-containing glycerolipids. After being pH 1-2 with 3 M methanolic HCl, the lipid was incubated at room temperature for 1 h in order to hydrolyze plasmalogens (22). Sphingomyelin and ceramide were separated by sequential one-dimensional thin-layer chromatography. Samples were spotted at 10 cm from bottom of 20 x 20 cm silica gel 60 plates and developed with chloroform/acetone (96: 4, v/v) to the top. After visualization of the spots with iodine vapor, the top portion of the plate was removed. The plate was cut at 3 mm below free fatty acid spots. Then, the plate was inverted and sequentially developed in the same direction with chloroform/benzene/ ethanol (80: 40 : 75, v/v) followed by chloroform/methanol!28 % ammonium hydroxide (65: 25: 5, v/v) (23). The areas corresponding to sphingomyelin and ceramide identified by co-migration with authentic standards, were scraped off the plates and the radioactivity was determined by a liquid scintillation counter (Beckmann LS 6500).

Results Effects of brefeldin A on PLD activity

RBL-2H3 cells were sensitized with anti-DNP monoclonal mouse IgE and then stimulated with DNP-Ascaris Ag (100 ng/ml). PLD catalyzes transphosphatidylation reaction leading to accumulation of phosphatidylalcohol in the presence of primary alcohol, which is a specific and metabolically

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INCUBATION TIME (min) Figure 1. Inhibition of Ag-induced PBut formation by BFA. RBL-2H3 cells were labeled with eH]palmitic acid for 20 h. After removal of unicorporated radiolabels, cells were incubated with or without 10 Ilg/ml BFA at 37°C for 20 min prior to stimulation. The cells were stimulated with 100 ng/ mJ Ag in the presence of 0.3 % butanol. After lipid extraction, phospholipids were separated on TLC plates. The radioactivity of eH]PBut was determined in a liquid scintillation counter. Details were described in Materials and Methods. 0: Control (stimulated buffer alone), . : Ag, &: Ag+ BFA. Data are means ± SD from two experiments each performed in duplicate. ". p < 0.05, "." P < 0.01 (t-test).

BFA inhibits Ag-induced PLD activity in RBL cells . 235

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Figure 2. Concentration-dependent effect of BFA on Ag-induced PBut formation. RBL2H3 cells labeled with eH]palmitic acid were preincubated with various concentrations of BFA for 20 min and then stimulated with 100 ng/ml Ag for 10 min. Lipid extraction and determination of radioactivity were performed as described in Materials and Methods. Each point is the mean of three experiments each performed in duplicate. Bars indicate SD. ". p < 0.05, ".". P < 0.01 (t-test).

stable product of PLD activation. Since butanol was a 10-fold more potent acceptor than ethanol for PLD activity (24), the formation of PBut was measured to monitor antigen-stimulated PLD activity in RBL-2H3 cells. When eH]palmitic acid-labeled RBL-2H3 cells were stimulated with Ag (100 ng/ml) in the presence of 0.3 % butanol, eH]PBut was rapidly produced at 2 min and reached the nearly plateau at 10 min (Fig. 1). eH]PBut increased from unstimulated control level of 0.15 % of total radioactivity to 0.36 % at 20 min after Ag addition. The maximal inhibition of PBut formation was obtained after 20 min preincubation of cells with BF A (data not shown). When the cells were preincubated with 10 /!g/ml (about 35 /!M) BFA for 20 min prior to stimulation, the formation of eH]PBut was inhibited (Fig. 1). BFA up to 20 /!g/ml had no effect on the basal eH]PBut level. However, the Ag-induced eH]PBut formation was inhibited in a concentration-dependent manner (Fig. 2). The inhibition was detectable at a BFA concentration of 1 /!g/ml and reached the maximum at 10 /!g/ml, where the Ag-induced PLD activation was inhibited by nearly 70 %. Further inhibition was not observed at a higher concentration (40 /!g/ . ml) of BFA (data not shown). We further examined whether BFA exerted the inhibitory effects on the receptor-bypass stimuli such as PMA and Ca2 + ionophore. PMA, a commonly used activator for PKC and ionophore A23187, caused PLD activation in RBL-2H3 cells, as previously observed (7). The eH]PBut formation

236 . Y.

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Figure 3. Effect of BFA on Ag-, A23187- or PMA-induced PBut formation. RBL-2H3 cells labeled with eH]palmitic acid were preincubated with 10 ~g/ml BFA for 20 min and were stimulated with 100 ng/ml Ag, 100 nM PMA or 500 nM A23187 for 10 min. Lipid extraction and determination of radioactivity were performed as described in Materials and Methods. Each point is the mean of three experiments each performed in duplicate. Bars indicate SD. 'f P < 0.05, ".". P < 0.01 (t-test).

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BFA(/l9/ml ) Figure 4. Concentration-dependent inhibitory effect of BFA on Ag-induced serotonin release. RBL-2H3 cells were labeled with [14C]5-HT for 16 h. After removing unincorporated radiolabels, cells were incubated with various concentrations of BFA for 20 min prior to stimulation. The cells were stimulated with Ag (100 ng/ml) for 30 min. 4 C]5HT release (%) was calculated as described in Materials and Methods. Each point is the mean of three experiments each performed in duplicate. Bars indicate SD. ". p < 0.05, ".". P < 0.01 (t-test).

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BFA inhibits Ag-induced PLD activity in RBL cells . 237

increased from 0.14 % (basal unstimulated level) to 0.75 % with PMA (100 nM) and to 0.91 % with A23187 (500 nM) (Fig. 3). When the cells were preincubated with BFA (10 f.tg/ml) for 20 min prior to stimulation, the A23187-induced eH]PBut formation was inhibited by 27 % (Fig. 3). In contrast, the PMA-induced eH]PBut formation was not affected by BFA (10 f.tg/ml). Since it has recently been demonstrated that BFA stimulated sphingomyelin biosynthesis or hydrolysis (25), leading to production of ceramide which in turn inhibits PLD activity in rat fibroblasts (26), the effects of BFA on sphingomyelin metabolism were examined in RBL-2H3 cells. When the cells were stimulated with Ag (100 ng/ml) for 10 min, the levels of ceramide and sphingomyelin were hardly changed (data not shown). Effects of brefeldin A on serotonin release

The secretory response was measured in [14C]5-HT-Iabeled RBL-2H3 cells, because the cells stored 5-HT in granules and released it in response to stimuli. BFA up to 10 f.tg/ml had no effect on spontaneous 4 C]5-HT release (data not shown). However, when RBL-2H3 cells were stimulated with Ag for 30 min, nearly 45 % of [14C]5-HT were released (data not shown). BFA suppressed Ag-induced 4 C]5-HT release in a concentrationdependent manner (Fig. 4). The inhibitory effect was evident at 0.2 f.tg/ml and reached the maximum at 0.5 f.tg/ml, a lower concentration than that

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Figure 5. Inhibition by BFA of Ag-, A23187- or PMA-induced serotonin release. RBL2H3 cells, labeled with 4 C]5-HT, were preincubated with or without 10 fig/ml BFA for 20 min and were stimulated with 100 ng/ml Ag, 100 nM PMA or 500 nM A23187 for 30 min. Release percent was calculated as described in Materials and Methods. Each point is the mean of three experiments each performed in duplicate. Bars indicate SD. ,:.,:. p < 0.01 (t-test).

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238 . Y.

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et al.

observed for PLD activation (10 Ilg/ml). In contrast, the maximal inhibition (at most 25 %) caused by 0.5 Ilg/ml was much lower than that observed for PLD activation (70 % inhibition). The inhibitory effects of BFA on PMAor A23187-induced secretory responses were further examined. A23187 (500 nM) caused [14C]5-HT release and 23 % of release was observed at 30 min after stimulation (Fig. 5). However, PMA (100 nM) failed to cause serotonin release. When the cells were preincubated with BFA (10 Ilg/ml) for 20 min before stimulation, A23187-induced 4C]5-HT release was inhibited (Fg. 5). The maximal inhibition (nearly 50 %) was much higher than that observed for A23187 -induced PLD activation.

ct

Discussion Several regulatory factors such as Ca2 +, PKC, GTP-binding proteins and protein tyrosine kinase have been proposed for PLD activation (19, 27). In RBL-2H3 cells, possible involvement of Ca2 + and PKC has been proposed (7, 9, 28). Our previous study suggested evidence that certain unidentified PTK would act between IgE-receptor and PLD (10). However, the detailed mechanism underlying the PLD activation is not fully disclosed. Recent reports (11-13) have proposed that ARF functions as an activator of PLD. In the cell-free assay of granulocytes, PLD activity was enhanced by guanosine 5' -O-(3-thiotriphosphate) (GTPyS), in the presence of both membranes and cytosol (29, 30). A cytosolic component that conferred sensitivity to GTPyS upon PLD activity was purified and identified as ARF (12). COCKCROFT'S group (13) also demonstrated that PLD was a downstream effector of ARF. ARF is known to act as a crucial factor for regulation of intracellular vesicle traffic (15) and BFA has been used to study membrane transport (16). ARF is active in the GTP-bound form and hydrolysis of bound GTP to GDP by ARF-GAP results in inactivation. This fungal product is considered to inhibit the GDP-GTP exchange of ARF, resulting in an incomplete assembly of coatomer onto the membrane vesicles (17, 18). Thus, this compound can be potentially used as an inhibitor for ARFmediated cellular events. Recently, STERNWEIS et al. (31) provided evidence that PLD activity in Golgi-enriched membrane prepared from Chinese hamster ovary cells was greatly stimulated by ARF and GTP analogs, and that this stimulation was effectively inhibited by BFA. These observations assessed the use of BFA as a potential inhibitor for ARF-mediated PLD activation. The results obtained in the present study demonstrated that BFA inhibited the Ag-induced PLD activation in RBL-2H3 cells. The inhibition by BFA was concentration-dependent and the maximal inhibition (nearly 70 %) was obtained at 10 Ilg/ml of the drug. However, further inhibition was not observed at its higher concentrations. BFA also exerted an inhibitory effect on the A23187 -induced PLD activation. In contrast, BFA failed

BFA inhibits Ag-induced PLD activity in RBL cells . 239

to inhibit the PMA-induced PLD activation. These data suggest the existence of at least two pathways; BFA -sensitive and -insensitive which lead to PLD activation. The BFA-sensitive PLD activation suggests the possible involvement of ARF in its regulation in RBL-2H3 cells. The BFA-insensitive pathway is probably mediated by PKC, since the PMA-induced PLD activation was not prevented by BFA. Evidence for the existence of PLD isozymes was recently provided by the recent reports (11, 32); one type is oleic acid-dependent and the other one is ARF-dependent. More recently, possible involvement of Rho family GTP binding proteins in PLD activation has been demonstrated in hepatocytes (33), neutrophils (34) and HL-60 cells (35, 36). In the in vitro assays, it is reasonable to consider that specific isoforms may be activated through different signaling pathways. It has recently been reported that BFA stimulates sphingomyelin biosynthesis in hepatocytes (37) or promotes sphingomyelin hydrolysis in HL-60 cells (29). In the latter case, cerami de is produced which inhibits PLD activity in rat fibroblasts (26). However, in RBL-2H3 cells BFA had no effect on sphingomyelin metabolism (data not shown). Thus, the inhibition of Ag-mediated PLD activation by BFA was not caused by the production of ceramide. LIN et al. (38) reported that in the presence of ethanol phosphatidylethanol production at the expense of P A reduced Ag-mediated histamine secretion in RBL-2H3 cells. Similar observations were obtained in chemoattractant-stimulated neutrophils (30, 39). These results suggest that PLD activation is closely coupled with degranulation reaction. However, the inhibitory profile of the Ag-mediated PLD activation by BFA was not closely correlated with that for serotonin secretion. BFA inhibited the Aginduced serotonin release at a concentration 20-fold lower than that needed for inhibition of PLD. In contrast, the maximal inhibition of the Agmediated PLD activation (70 %) by BFA was greater than that of the secretory response (25 %). Furthermore, PMA caused significant elevation of PLD activation, however, no increase in serotonin release. Thus, these results do not support the view that PLD is an important signaling enzyme for secretory responses, although several lines of evidence suggest that PLD activation is involved in exocytosis. In summary, the data obtained in the present study indicate that there are at least two Ag-stimulated PLD signaling pathways: BFA-sensitive and -insensitive PLD activation in RBL-2H3 cells. PKC plays a role in BFAinsensitive pathway. Further, it was suggested that ARF plays a role in BFA-sensitive PLD activation in RBL-2H3 cells. More works including the reconstitution system are required to elucidate the role of ARF in PLD activation in RBL-2H3 cells and are under current progress in our laboratory . Acknowledgement

This study was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.

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