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Effect ofClostridium difficileToxin B on IgE Receptor-Mediated Signal Transduction in Rat Basophilic Leukemia Cells: Inhibition of Phospholipase D Activation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
224, 591–596 (1996)
1069
Effect of Clostridium difficile Toxin B on IgE Receptor-Mediated Signal Transduction in Rat Basophilic Leukemia Cells: Inhibition of Phospholipase D Activation Katsuhiro Ojio,*,1 Yoshiko Banno,† Shigeru Nakashima,† Naoki Kato,‡ Kunitomo Watanabe,‡ David M. Lyerly,§ Hideo Miyata,* and Yoshinori Nozawa† *Department of Otolaryngology and †Department of Biochemistry and ‡Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan; and §TechLab, Inc., VPI Corporate Research Center, 1861 Pratt Drive, Blacksburg, Virginia 24060-6371 Received June 13, 1996 Antigen (Ag)-stimulated phospholipase D (PLD) activation and secretion were almost abolished by pretreatment of rat basophilic leukemia (RBL)-2H3 cells for 4 h with 5 ng/ml Clostridium difficile Toxin B which is known to inhibit Rho family proteins (Rho, Cdc42, Rac). The concentration-dependent inhibition of PLD activation was well correlated with the level of glucosylation of Rho family proteins. In streptolysin O-permeabilized RBL cells, Toxin B suppressed [3H] phosphatidylbutanol (PBut) formation in response to guanosine 5*-O-(3-thiotriphosphate) (GTPgS) and phorbol 12-myristate 13-acetate (PMA) by 67 and 43%, respectively. The synergistic PLD activation by GTPgS and PMA was also reduced by Toxin B by 67%. These results suggest that the IgE receptor-coupled PLD activation is largely mediated by Rho proteins. q 1996 Academic Press, Inc.
Rat basophilic leukemia (RBL)-2H3 cells share many of the properties of mucosal mast cells and express high affinity receptor for IgE (FceRI). Aggregation of FceRI leads to activation of various signal transducing phospholipases including phospholipase D (PLD) and release of chemical mediators (1-3). Several factors, such as protein kinase C (PKC), Ca2/, GTP-binding proteins, oleic acid, and PIP2 are proposed to be involved in the regulation of PLD activation in various cells (4). We have previously suggested the possible involvement of protein tyrosine kinase (5), ADP-ribosylation factor (ARF) (6) and calcium/calmodulin (7) in antigen (Ag)stimulated PLD activation in RBL-2H3 cells. However, the regulatory mechanisms of PLD activation are still poorly understood. In addition to ARF, the involvement of Rho family proteins in PLD activation has been suggested in neutrophils (8), HL60 cells (9, 10), and hepatocytes (11). However, their roles in PLD activation in RBL-2H3 cells have not yet been investigated. Recently, Just et al. (12) have shown evidence that the potent cytotoxins from Clostridium difficile (C. difficile), Toxin A and B are monoglucosyltransferases which catalyze the incorporation of glucose into threonine residue at position 37 of Rho family proteins, and that glucosylation by these toxins may inhibit their interactions with effector molecules. In fact, this modification prevents the ability of Rho family proteins to induce polymerization of actin filaments (13). These findings indicate that the toxins can be used as useful tools to analyze the functions of Rho family proteins. In the present study, we have examined the role 1
To whom correspondence should be addressed. Fax: (81)58-262-2461. Abbreviations: PLD, phospholipase D; GTPgS, guanosine 5*-O-(3-thiotri-phosphate); PMA, phorbol 12-myristate 13-acetate; RBL, rat basophilic leukemia; PKC, protein kinase C; Ag, antigen; C. difficile, Clostridium difficile; SLO, streptolysin O; PBut, phosphatidylbutanol; SDS-PAGE, sodium dodecylsulfate polyacrylamide-gel electrophoresis; ARF, ADP-ribosylation factor, FceRI; high affinity receptor for IgE; PIP2, phosphatidylinositol 4,5-bisphosphate; DNP, dinitrophenol. 591 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of Rho family proteins, using the C. difficile toxins, in IgE receptor-mediated PLD activation in RBL-2H3 cells. MATERIALS AND METHODS Materials. RBL-2H3 cells were obtained from Health Science Research Resources. Cells were cultured as previously described (14). Toxin A and Toxin B were extracted and purified from C. difficile. Monoclonal mouse anti-dinitrophenyl (DNP) IgE was obtained from Seikagaku Kogyo. [14C]5-Hydroxytryptamine binoxalate (serotonin, 5-HT) (50 mCi/ mmol) and Aquasol-2 were purchased from Dupont-New England Nuclear. [9,10-3H] Palmitic acid (52.4 Ci/mmol), UDP-[U-14C]glucose (302 mCi/mmol), ECL and anti-rabbit IgG horseradish peroxidase-coupled secondary antibodies were from Amersham International. Streptolysin O (SLO) was from Eiken Chemical Co. Guanosine 5*-O-(3-thiotriphosphate) (GTPgS) was from Boehringer Mannheim. Antibodies against small GTP-binding proteins (RhoA, Rac1, Rac2, Cdc42Hs) and PKC isozymes were from Santa Cruz Biotechnology. Serotonin release. Serotonin release was measured using [14C]serotonin-prelabeled cells according to the method of Nakamura et al (14). Assay of PLD activity in intact and permeabilized cells. RBL-2H3 cells (1.21106 cells/35-mm dishes) were incubated with [3H]palmitic acid (2 mCi/dish) and anti-DNP IgE (0.3 mg/ml) for 20 h. Toxin A or Toxin B was added for the last indicated time of radiolabeling. After removal of the radiolabeling medium, the dishes were rinsed three times and equilibrated in Tyrode-HEPES solution (NaCl 134 mM, NaHCO3 12 mM, KCl 2.9 mM, MgCl2 1 mM, CaCl2 1.8 mM, NaH2PO4 0.36 mM, glucose 5.6 mM, HEPES 10 mM, 0.1% BSA, pH7.4) for 5 min at 377C. Cells were then stimulated for 20 min with 100 ng/ml DNP-Ascaris antigen. In order to examine the effect of GTPgS, cells were permeabilized with 1.2 units/ml SLO for 5 min in PIPES-buffered solution (potassium glutamate 138 mM, glucose 5 mM, PIPES 20 mM, ATP 5 mM; magnesium acetate 7 mM, EGTA 1 mM, and CaCl2 670 mM to give a calculated concentration of free Ca2/ of 1 mM, pH7.0) (15) and then incubated with phorbol 12-myristate 13-acetate (PMA) and/or GTPgS. The activity of PLD was assessed by the formation of [3H]phosphatidylbutanol (PBut) in the presence of 0.3% butanol. After lipid extraction, [3H]PBut was separated by one dimensional thin-layer chromatography (16). The area corresponding to [3H]PBut, identified by co-migration with PBut standard, was scrapped off the plate and the radioactivity was determined in a liquid scintillation counter (Beckman LS 6500). Assay of glucosylation activity. Glucosylation of proteins in RBL-2H3 cell lysates was measured by the method described by Just et al. (12), with minor modifications. Cells were pretreated with Toxin A or Toxin B for 4 h, and lysed by sonication. Cell lysates (1 mg protein /ml) were incubated with 10 mg/ml Toxin A or 1 mg/ml Toxin B in the presence of 30 mM UDP-[14C]glucose at 377C for 20 min. Aliquots mixed with Laemmli’s sample buffer were subjected to electrophoresis on 13% polyacrylamide gel. Incorporation of UDP-[14C]glucose was analyzed by a
FIG. 1. Inhibitory effect of Toxin B on Ag-induced PLD activation and secretion in RBL-2H3 cells. RBL-2H3 cells were preincubated with [3H]palmitic acid and anti-DNP IgE for 20 h. Cells were incubated with indicated concentrations of Toxin B for 4 h (A and C) or with 5 ng/ml of Toxin B for indicated periods of time (B). PLD activity (A and B) and [14C]serotonin release (C) were measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate. 592
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FIG. 2. [14C]UDP-glucosylation of Rho family proteins in RBL-2H3 cells. (A) Western blot analysis of Rho family proteins. Proteins were separated by SDS-PAGE on 13% polyacrylamide gels and electrophoretically transferred onto nitrocellulose membrane. Western blot analysis using specific antibodies was performed as described under ‘‘Materials and Methods’’. (B) UDP-[14C]glucose incorporation into Mr Ç22K protein. RBL-2H3 cells were pre-treated with indicated concentrations of Toxin A (h) or Toxin B (s) for 4 h. After cell lysis, incorporation of UDP-[14C]glucose was quantified as described under the ‘‘Materials and Methods’’. Data are expressed as relative percent of the untreated control (100%).
bioimaging analyzer (FUJIX BAS 2000, Fuji). Density of each band was quantified by densitometer (Densitograph for the Macintosh ver.3.0, ATTO). Electrophoresis and Western blot analysis. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (17) on 13 and 8% polyacrylamide gels for small GTP-binding proteins and PKCs, respectively. Proteins were then electrophoretically transferred onto nitrocellulose membrane. Western blot analysis using specific antibodies was performed as previously described (14).
RESULTS AND DISCUSSION
Rho family GTP-binding proteins (Rho, Rac, Cdc42) are thought to be involved in the regulation of actin cytoskeleton (13, 18–20). Moreover, these proteins, especially RhoA, have been reported to play a role in the regulation of PLD activity (8–11). C. difficile Toxin B, which specifically glucosylates these proteins and inhibits their interactions with effectors, is a useful tool to study the functions of these proteins. In human embryonik kidney cells (21), Toxin B was shown to inhibit carbachol-stimulated PLD activation. Furthermore, recent report (22) has demonstrated that Toxin B inhibited the Ag-induced serotonin release in RBL-2H3 cells, suggesting involvement of Rho proteins in Ag-induced signal transduction. To examine their role in Ag-mediated PLD activation in RBL-2H3 cells, the cells were treated with C. difficile Toxin B. It was shown that the PLD activation by Ag was inhibited by Toxin B in a concentration-dependent manner (Fig. 1A). After treatment of cells for 4 h, more than 2 ng/ ml of the toxin markedly suppressed PLD activation and a maximal inhibition (more than 593
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FIG. 3. GTPgS-induced PLD activation in permeabilized RBL-2H3 cells. RBL-2H3 cells were prelabeled with [3H]palmitic acid for 20 h. Cells were then permeabilized with 1.2 units/ml SLO for 5 min and stimulated with 30 mM GTPgS for the indicated times in the presence of 0.3% butanol. PLD activity was measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate.
80%) was obtained at 5 ng/ml. The inhibitory effect was dependent on preincubation time (Fig. 1B). With 5 ng/ml Toxin B, marked inhibition was observed after pretreatment of cells for more than 2 h, and at the 3 h-pretreatment the Ag-mediated PLD activation was suppressed by 80%. However, Toxin A failed to suppress PLD activation even at 100 ng/ml after the 4 h-pretreatment (data not shown). As previously shown (22), Toxin B (10 ng/ml) inhibited Aginduced serotonin release maximally by about 80% after 4 h-pretreatment of RBL-2H3 cells (Fig. 1C). The inhibitory profile by Toxin B in Ag-induced PLD activation was consistent with that in serotonin release. These data are compatible with the previous notion that PLD is implicated in the secretory response (23). Under the same conditions in Fig. 1A, we examined whether Rho proteins in RBL-2H3 cells were glucosylated by Toxin B treatment. In RBL-2H3 cells, RhoA and Cdc42 are rich but much less in Rac1 as inferred by immunoblotting (Fig. 2A). As shown in Fig. 2B, Toxin B induced incorporation of UDP-[14C]glucose into the protein band with Mr Ç22 K which cross-reacted with anti-RhoA, -Cdc42 and -Rac1 antibodies. However, these Rho proteins could not be separated as single bands by our SDS-PAGE. Therefore, Toxin B-catalyzed incorporation of UDP-[14C]glucose into the proteins was determined in lysates of RBL-2H3 cells pretreated with or without Toxin B. The pretreatment of Toxin B reduced dose-dependently the incorporation of UDP-[14C]glucose into the Mr Ç22 K proteins, indicating that endogenous Rho family proteins were glucosylated by the prior Toxin B treatment (Fig. 2B). Toxin B (10 ng/ml) glucosylated 80% of the proteins by 4 h-pretreatment. In contrast, Toxin A was without effect on glucosylation in the same condition. The level of glucosylation of the Rho proteins by Toxin B was correlated with that of inhibition of Ag-stimulated PLD activation, suggesting an involvement of Rho proteins in IgE receptor-mediated PLD activation in RBL-2H3 cells. Previous studies have shown that in permeabilized RBL-2H3 cells, secretion can be stimulated by GTPgS alone or plus phorbol ester PMA which activates PKCa and b (24, 25). Our recent study has indicated that RhoA and PKCa synergistically activated a membrane-associated PLD in HL60 cells (9). In the current study with SLO-permeabilized RBL-2H3 cells, [3H]PBut formation in response to GTPgS (30 mM) was increased with times up to 60 min (Fig. 3). At 60 min-incubation, Western blot analysis revealed that half of RhoA and nearly all of PKCa remained in the permeabilized cells (data not shown). [3H]PBut formation during 30 min incubation with 30 mM GTPgS or 100 nM PMA was 1.75% and 0.86% of total radioactivity, respectively. The 4 h-pretreatment with Toxin B suppressed [3H]PBut formation 594
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FIG. 4. Inhibitory effect of Toxin B on GTPgS- or PMA-induced PLD activation in permeabilized RBL-2H3 cells. RBL-2H3 cells were preincubated with [3H]palmitic acid for 20 h and with 5 ng/ml Toxin B for the last 4 h. Cells were permeabilized with 1.2 units/ml SLO for 5 min and stimulated with 100 nM PMA and/or 30 mM GTPgS for 30 min in the presence of 0.3% butanol. PLD activity was measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate.
induced by GTPgS or PMA by 67 and 43%, respectively (Fig. 4). PMA and GTPgS exerted a synergistic activation of PLD. Toxin B inhibited this synergistic enhancement in [3H]PBut formation by 67%. These results suggest that GTPgS-induced PLD activation was largely dependent on Rho proteins. The PLD activation insensitive to Toxin B was probably dependent on other GTP-binding protein(s). ADP-ribosylation factor (ARF) is one of the most likely candidates; an ARF inhibitor, brefeldin A inhibited Ag-induced PLD activation (6). The Toxin B-treatment exerted an equivalent inhibition of Ag-induced serotonin release and PLD activation by glucosylation of Rho proteins (Figs. 1 and 2), leading us to speculate that Rho proteinsmediated PLD activation could be somehow associated with secretory process. The in vitro experiments have demonstrated that members of Rho protein family are differentially implicated in PLD activation dependent on type of cells; RhoA, Rac1 and Cdc42 in HL60 cells (10), RhoA and Rac1 in rat liver (11), and RhoA and Cdc42 in porcine brain (26). In intact RBL-2H3 cells, it was recently shown that Cdc42 rather than RhoA takes a greater part in Ag-induced secretion (22). However, the relative contribution of RhoA and Cdc42 in IgE receptor-mediated PLD activation remains to be determined. ACKNOWLEDGMENT This work was supported in part by research grants from the Ministry of Education, Culture and Science of Japan.
REFERENCES 1. Beaven, M. A., Moore, J. P., Smith, G. A., Hesketh, T. B., and Metcalfe, J. C. (1984) J. Biol. Chem. 259, 7137– 7242. 2. Maeyama, K., Hohman, R. J., Ali, H., Cunha-Melo, J. R., and Beaven, M. A. (1988) J. Immunol. 140, 3919– 3927. 3. Ishizuka, Y., Imai, A., and Nozawa, Y. (1984) Biochem. Biophys. Res. Commum. 123, 875–881. 4. Liscovitch, M., and Chalifa, V. (1994) in Signal-Activated Phospholipases (M. Liscovitch, Ed.), pp. 32–63. R. G. Landes, Austin, TX. 5. Kumada, T., Miyata, H., and Nozawa, Y. (1993) Biochem. Biophys. Res. Commum. 191, 1363–1368. 6. Nakamura, Y., Nakashima, S., Kumada, T., Ojio, K., Miyata, H., and Nozawa, Y. Immunobiology, in press. 7. Kumada, T., Nakashima, S., Nakamura, Y., Miyata, H., and Nozawa, Y. (1995) Biochim. Biophys. Acta. 1258, 107–114. 595
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8. Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509–21512. 9. Ohguchi, K., Banno, Y., Nakashima, S., and Nozawa, Y. (1996) J. Biol. Chem. 271, 4366–4372. 10. Siddiqi, A. R., Smith, J. L., Ross, A. H., Qiu, R. G., Symons, M., and Exton, J. H. (1995) J. Biol. Chem. 270, 8466–8473. 11. Malcolm, K. C., Ross, A. H., Qiu, R. G., Symons, M., and Exton, J. H. (1994) J. Biol. Chem. 269, 25951–25954. 12. Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500–503. 13. Aktories, K., and Just, I. (1995) Trends Cell Biol. 5, 441–443. 14. Nakamura, Y., Nakashima, S., Ojio, K., Banno, Y., Miyata, H., and Nozawa, Y. (1996) J. Immunol. 156, 256– 262. 15. Cunham-Melo, J. R., Gonzaga, H. M., Ali, H., Huang, F. L., Huang, K. P., and Beaven, M. A. (1989) J. Immunol. 143, 2617–2625. 16. Liscovitch, M., and Amsterdam, A. (1989) J. Biol. Chem. 264, 11762–11767. 17. Laemmli, U. K. (1970) Nature 227, 680–685. 18. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401–410. 19. Ridley, A. J., and Hall, A. (1992) Cold Spring Harb. Sym. 57, 661–671. 20. Norman, J. C., Price, L. S., Ridley, A. J., Hall, A., and Koffer, A. (1994) J. Cell Biol. 126, 1005–1015. 21. Schimidt, M., Rumennapp, U., Bienek, C., Keller, J., von Eichel-Streiber, C., and Jakobs, K. H. (1996) J. Biol. Chem. 271, 2422–2426. 22. Prepens, U., Just, I., von Eichel-Streiber, C., and Aktories, K. (1996) J. Biol. Chem. 271, 7324–7329. 23. Lin, P. Y., Wiggan, G. A., and Gilfillan, A. M. (1991) J. Immunol. 146, 1609–1614. 24. Buccione, R., Di Tullio, G. D., Caretta, M., Marinetti, M. R., Bizzarri, C., Francavilla, S., Luini, A., and De Matteis, A. (1994) Biochem. J. 298, 149–156. 25. Sagi-Eisenberg (1989) Trends Biochem. Sci. 14, 355–357. 26. Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944–14950.
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Effect of Clostridium difficile Toxin B on IgE Receptor-Mediated Signal Transduction in Rat Basophilic Leukemia Cells: Inhibition of Phospholipase D Activation Katsuhiro Ojio,*,1 Yoshiko Banno,† Shigeru Nakashima,† Naoki Kato,‡ Kunitomo Watanabe,‡ David M. Lyerly,§ Hideo Miyata,* and Yoshinori Nozawa† *Department of Otolaryngology and †Department of Biochemistry and ‡Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan; and §TechLab, Inc., VPI Corporate Research Center, 1861 Pratt Drive, Blacksburg, Virginia 24060-6371 Received June 13, 1996 Antigen (Ag)-stimulated phospholipase D (PLD) activation and secretion were almost abolished by pretreatment of rat basophilic leukemia (RBL)-2H3 cells for 4 h with 5 ng/ml Clostridium difficile Toxin B which is known to inhibit Rho family proteins (Rho, Cdc42, Rac). The concentration-dependent inhibition of PLD activation was well correlated with the level of glucosylation of Rho family proteins. In streptolysin O-permeabilized RBL cells, Toxin B suppressed [3H] phosphatidylbutanol (PBut) formation in response to guanosine 5*-O-(3-thiotriphosphate) (GTPgS) and phorbol 12-myristate 13-acetate (PMA) by 67 and 43%, respectively. The synergistic PLD activation by GTPgS and PMA was also reduced by Toxin B by 67%. These results suggest that the IgE receptor-coupled PLD activation is largely mediated by Rho proteins. q 1996 Academic Press, Inc.
Rat basophilic leukemia (RBL)-2H3 cells share many of the properties of mucosal mast cells and express high affinity receptor for IgE (FceRI). Aggregation of FceRI leads to activation of various signal transducing phospholipases including phospholipase D (PLD) and release of chemical mediators (1-3). Several factors, such as protein kinase C (PKC), Ca2/, GTP-binding proteins, oleic acid, and PIP2 are proposed to be involved in the regulation of PLD activation in various cells (4). We have previously suggested the possible involvement of protein tyrosine kinase (5), ADP-ribosylation factor (ARF) (6) and calcium/calmodulin (7) in antigen (Ag)stimulated PLD activation in RBL-2H3 cells. However, the regulatory mechanisms of PLD activation are still poorly understood. In addition to ARF, the involvement of Rho family proteins in PLD activation has been suggested in neutrophils (8), HL60 cells (9, 10), and hepatocytes (11). However, their roles in PLD activation in RBL-2H3 cells have not yet been investigated. Recently, Just et al. (12) have shown evidence that the potent cytotoxins from Clostridium difficile (C. difficile), Toxin A and B are monoglucosyltransferases which catalyze the incorporation of glucose into threonine residue at position 37 of Rho family proteins, and that glucosylation by these toxins may inhibit their interactions with effector molecules. In fact, this modification prevents the ability of Rho family proteins to induce polymerization of actin filaments (13). These findings indicate that the toxins can be used as useful tools to analyze the functions of Rho family proteins. In the present study, we have examined the role 1
To whom correspondence should be addressed. Fax: (81)58-262-2461. Abbreviations: PLD, phospholipase D; GTPgS, guanosine 5*-O-(3-thiotri-phosphate); PMA, phorbol 12-myristate 13-acetate; RBL, rat basophilic leukemia; PKC, protein kinase C; Ag, antigen; C. difficile, Clostridium difficile; SLO, streptolysin O; PBut, phosphatidylbutanol; SDS-PAGE, sodium dodecylsulfate polyacrylamide-gel electrophoresis; ARF, ADP-ribosylation factor, FceRI; high affinity receptor for IgE; PIP2, phosphatidylinositol 4,5-bisphosphate; DNP, dinitrophenol. 591 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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of Rho family proteins, using the C. difficile toxins, in IgE receptor-mediated PLD activation in RBL-2H3 cells. MATERIALS AND METHODS Materials. RBL-2H3 cells were obtained from Health Science Research Resources. Cells were cultured as previously described (14). Toxin A and Toxin B were extracted and purified from C. difficile. Monoclonal mouse anti-dinitrophenyl (DNP) IgE was obtained from Seikagaku Kogyo. [14C]5-Hydroxytryptamine binoxalate (serotonin, 5-HT) (50 mCi/ mmol) and Aquasol-2 were purchased from Dupont-New England Nuclear. [9,10-3H] Palmitic acid (52.4 Ci/mmol), UDP-[U-14C]glucose (302 mCi/mmol), ECL and anti-rabbit IgG horseradish peroxidase-coupled secondary antibodies were from Amersham International. Streptolysin O (SLO) was from Eiken Chemical Co. Guanosine 5*-O-(3-thiotriphosphate) (GTPgS) was from Boehringer Mannheim. Antibodies against small GTP-binding proteins (RhoA, Rac1, Rac2, Cdc42Hs) and PKC isozymes were from Santa Cruz Biotechnology. Serotonin release. Serotonin release was measured using [14C]serotonin-prelabeled cells according to the method of Nakamura et al (14). Assay of PLD activity in intact and permeabilized cells. RBL-2H3 cells (1.21106 cells/35-mm dishes) were incubated with [3H]palmitic acid (2 mCi/dish) and anti-DNP IgE (0.3 mg/ml) for 20 h. Toxin A or Toxin B was added for the last indicated time of radiolabeling. After removal of the radiolabeling medium, the dishes were rinsed three times and equilibrated in Tyrode-HEPES solution (NaCl 134 mM, NaHCO3 12 mM, KCl 2.9 mM, MgCl2 1 mM, CaCl2 1.8 mM, NaH2PO4 0.36 mM, glucose 5.6 mM, HEPES 10 mM, 0.1% BSA, pH7.4) for 5 min at 377C. Cells were then stimulated for 20 min with 100 ng/ml DNP-Ascaris antigen. In order to examine the effect of GTPgS, cells were permeabilized with 1.2 units/ml SLO for 5 min in PIPES-buffered solution (potassium glutamate 138 mM, glucose 5 mM, PIPES 20 mM, ATP 5 mM; magnesium acetate 7 mM, EGTA 1 mM, and CaCl2 670 mM to give a calculated concentration of free Ca2/ of 1 mM, pH7.0) (15) and then incubated with phorbol 12-myristate 13-acetate (PMA) and/or GTPgS. The activity of PLD was assessed by the formation of [3H]phosphatidylbutanol (PBut) in the presence of 0.3% butanol. After lipid extraction, [3H]PBut was separated by one dimensional thin-layer chromatography (16). The area corresponding to [3H]PBut, identified by co-migration with PBut standard, was scrapped off the plate and the radioactivity was determined in a liquid scintillation counter (Beckman LS 6500). Assay of glucosylation activity. Glucosylation of proteins in RBL-2H3 cell lysates was measured by the method described by Just et al. (12), with minor modifications. Cells were pretreated with Toxin A or Toxin B for 4 h, and lysed by sonication. Cell lysates (1 mg protein /ml) were incubated with 10 mg/ml Toxin A or 1 mg/ml Toxin B in the presence of 30 mM UDP-[14C]glucose at 377C for 20 min. Aliquots mixed with Laemmli’s sample buffer were subjected to electrophoresis on 13% polyacrylamide gel. Incorporation of UDP-[14C]glucose was analyzed by a
FIG. 1. Inhibitory effect of Toxin B on Ag-induced PLD activation and secretion in RBL-2H3 cells. RBL-2H3 cells were preincubated with [3H]palmitic acid and anti-DNP IgE for 20 h. Cells were incubated with indicated concentrations of Toxin B for 4 h (A and C) or with 5 ng/ml of Toxin B for indicated periods of time (B). PLD activity (A and B) and [14C]serotonin release (C) were measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate. 592
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FIG. 2. [14C]UDP-glucosylation of Rho family proteins in RBL-2H3 cells. (A) Western blot analysis of Rho family proteins. Proteins were separated by SDS-PAGE on 13% polyacrylamide gels and electrophoretically transferred onto nitrocellulose membrane. Western blot analysis using specific antibodies was performed as described under ‘‘Materials and Methods’’. (B) UDP-[14C]glucose incorporation into Mr Ç22K protein. RBL-2H3 cells were pre-treated with indicated concentrations of Toxin A (h) or Toxin B (s) for 4 h. After cell lysis, incorporation of UDP-[14C]glucose was quantified as described under the ‘‘Materials and Methods’’. Data are expressed as relative percent of the untreated control (100%).
bioimaging analyzer (FUJIX BAS 2000, Fuji). Density of each band was quantified by densitometer (Densitograph for the Macintosh ver.3.0, ATTO). Electrophoresis and Western blot analysis. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (17) on 13 and 8% polyacrylamide gels for small GTP-binding proteins and PKCs, respectively. Proteins were then electrophoretically transferred onto nitrocellulose membrane. Western blot analysis using specific antibodies was performed as previously described (14).
RESULTS AND DISCUSSION
Rho family GTP-binding proteins (Rho, Rac, Cdc42) are thought to be involved in the regulation of actin cytoskeleton (13, 18–20). Moreover, these proteins, especially RhoA, have been reported to play a role in the regulation of PLD activity (8–11). C. difficile Toxin B, which specifically glucosylates these proteins and inhibits their interactions with effectors, is a useful tool to study the functions of these proteins. In human embryonik kidney cells (21), Toxin B was shown to inhibit carbachol-stimulated PLD activation. Furthermore, recent report (22) has demonstrated that Toxin B inhibited the Ag-induced serotonin release in RBL-2H3 cells, suggesting involvement of Rho proteins in Ag-induced signal transduction. To examine their role in Ag-mediated PLD activation in RBL-2H3 cells, the cells were treated with C. difficile Toxin B. It was shown that the PLD activation by Ag was inhibited by Toxin B in a concentration-dependent manner (Fig. 1A). After treatment of cells for 4 h, more than 2 ng/ ml of the toxin markedly suppressed PLD activation and a maximal inhibition (more than 593
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FIG. 3. GTPgS-induced PLD activation in permeabilized RBL-2H3 cells. RBL-2H3 cells were prelabeled with [3H]palmitic acid for 20 h. Cells were then permeabilized with 1.2 units/ml SLO for 5 min and stimulated with 30 mM GTPgS for the indicated times in the presence of 0.3% butanol. PLD activity was measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate.
80%) was obtained at 5 ng/ml. The inhibitory effect was dependent on preincubation time (Fig. 1B). With 5 ng/ml Toxin B, marked inhibition was observed after pretreatment of cells for more than 2 h, and at the 3 h-pretreatment the Ag-mediated PLD activation was suppressed by 80%. However, Toxin A failed to suppress PLD activation even at 100 ng/ml after the 4 h-pretreatment (data not shown). As previously shown (22), Toxin B (10 ng/ml) inhibited Aginduced serotonin release maximally by about 80% after 4 h-pretreatment of RBL-2H3 cells (Fig. 1C). The inhibitory profile by Toxin B in Ag-induced PLD activation was consistent with that in serotonin release. These data are compatible with the previous notion that PLD is implicated in the secretory response (23). Under the same conditions in Fig. 1A, we examined whether Rho proteins in RBL-2H3 cells were glucosylated by Toxin B treatment. In RBL-2H3 cells, RhoA and Cdc42 are rich but much less in Rac1 as inferred by immunoblotting (Fig. 2A). As shown in Fig. 2B, Toxin B induced incorporation of UDP-[14C]glucose into the protein band with Mr Ç22 K which cross-reacted with anti-RhoA, -Cdc42 and -Rac1 antibodies. However, these Rho proteins could not be separated as single bands by our SDS-PAGE. Therefore, Toxin B-catalyzed incorporation of UDP-[14C]glucose into the proteins was determined in lysates of RBL-2H3 cells pretreated with or without Toxin B. The pretreatment of Toxin B reduced dose-dependently the incorporation of UDP-[14C]glucose into the Mr Ç22 K proteins, indicating that endogenous Rho family proteins were glucosylated by the prior Toxin B treatment (Fig. 2B). Toxin B (10 ng/ml) glucosylated 80% of the proteins by 4 h-pretreatment. In contrast, Toxin A was without effect on glucosylation in the same condition. The level of glucosylation of the Rho proteins by Toxin B was correlated with that of inhibition of Ag-stimulated PLD activation, suggesting an involvement of Rho proteins in IgE receptor-mediated PLD activation in RBL-2H3 cells. Previous studies have shown that in permeabilized RBL-2H3 cells, secretion can be stimulated by GTPgS alone or plus phorbol ester PMA which activates PKCa and b (24, 25). Our recent study has indicated that RhoA and PKCa synergistically activated a membrane-associated PLD in HL60 cells (9). In the current study with SLO-permeabilized RBL-2H3 cells, [3H]PBut formation in response to GTPgS (30 mM) was increased with times up to 60 min (Fig. 3). At 60 min-incubation, Western blot analysis revealed that half of RhoA and nearly all of PKCa remained in the permeabilized cells (data not shown). [3H]PBut formation during 30 min incubation with 30 mM GTPgS or 100 nM PMA was 1.75% and 0.86% of total radioactivity, respectively. The 4 h-pretreatment with Toxin B suppressed [3H]PBut formation 594
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FIG. 4. Inhibitory effect of Toxin B on GTPgS- or PMA-induced PLD activation in permeabilized RBL-2H3 cells. RBL-2H3 cells were preincubated with [3H]palmitic acid for 20 h and with 5 ng/ml Toxin B for the last 4 h. Cells were permeabilized with 1.2 units/ml SLO for 5 min and stimulated with 100 nM PMA and/or 30 mM GTPgS for 30 min in the presence of 0.3% butanol. PLD activity was measured as described under ‘‘Materials and Methods’’. Data are means { SD of two separate experiments each performed in duplicate.
induced by GTPgS or PMA by 67 and 43%, respectively (Fig. 4). PMA and GTPgS exerted a synergistic activation of PLD. Toxin B inhibited this synergistic enhancement in [3H]PBut formation by 67%. These results suggest that GTPgS-induced PLD activation was largely dependent on Rho proteins. The PLD activation insensitive to Toxin B was probably dependent on other GTP-binding protein(s). ADP-ribosylation factor (ARF) is one of the most likely candidates; an ARF inhibitor, brefeldin A inhibited Ag-induced PLD activation (6). The Toxin B-treatment exerted an equivalent inhibition of Ag-induced serotonin release and PLD activation by glucosylation of Rho proteins (Figs. 1 and 2), leading us to speculate that Rho proteinsmediated PLD activation could be somehow associated with secretory process. The in vitro experiments have demonstrated that members of Rho protein family are differentially implicated in PLD activation dependent on type of cells; RhoA, Rac1 and Cdc42 in HL60 cells (10), RhoA and Rac1 in rat liver (11), and RhoA and Cdc42 in porcine brain (26). In intact RBL-2H3 cells, it was recently shown that Cdc42 rather than RhoA takes a greater part in Ag-induced secretion (22). However, the relative contribution of RhoA and Cdc42 in IgE receptor-mediated PLD activation remains to be determined. ACKNOWLEDGMENT This work was supported in part by research grants from the Ministry of Education, Culture and Science of Japan.
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