©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of FcRI-mediated Activation of Rat Basophilic Leukemia Cells by Clostridium difficile Toxin B (Monoglucosyltransferase) (*)

(Received for publication, November 1, 1995; and in revised form, December 27, 1995)

Ulrike Prepens Ingo Just Christoph von Eichel-Streiber (1) Klaus Aktories (§)

From the Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg Institut für Medizinische Mikrobiologie und Hygiene der Johannes-Gutenberg-Universität, D-55101 Mainz, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [^3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B ^14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 µg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [^3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.


INTRODUCTION

Activation of rat basophilic leukemia (RBL) (^1)2H3-hm1 cells by antigen depends on cross-linking of IgE-receptor complexes and a cascade of subsequent biochemical reactions that finally result in secretion of histamine, serotonin, and other inflammatory substances (for review, see (1) ). So far the precise signal transduction pathway is not clear. As deduced from the data obtained in several laboratories, FcRI stimulation appears to activate tyrosine kinases (2, 3, 4, 5, 6) and phospholipase C (2, 7, 8, 9) with subsequent mobilization of Ca and protein kinase C and may further involve phospholipase D (10) and/or phospholipase A(2)(11, 12) activation. It has been suggested that GTP-binding proteins are involved in the activation cascade because GTPS induces secretion in permeabilized mast cells(13, 14) . This is particular true for the activation of peritoneal mast cells by compound 48/80, which is blocked by pertussis toxin(15, 16) . However, pertussis toxin has apparently no effect on antigen-stimulated mast cell activation(16) . Furthermore, some evidence has been presented for the involvement of a GTP-binding protein in the late step of secretion (13, 14) .

Low molecular mass GTP-binding proteins of the Rho subfamily (RhoA, RhoB, RhoC, Rac1, 2, Cdc42Hs) are involved in the regulation of the actin cytoskeleton(17) . Rho regulates growth factor-induced formation of stress fibers (18, 19) and formation of cell adhesions(20) . Rac is involved in membrane ruffling and lamellipodia(17, 21) , and Cdc42 participates in receptor-induced formation of microspikes(21) . Furthermore, Rho subfamily proteins appear to be molecular switches in various signal transduction pathways. They participate in cell-cell contact(22) , sensitization of smooth muscle contraction toward Ca(23) , and are involved in phospholipase D (24) and phosphatidylinositol-4-phosphate-5-kinase (25) regulation. Rho subfamily proteins are suggested to be involved in signal transduction upstream (26) or downstream (27) from phosphatidylinositol 3-kinase. Moreover, it was shown very recently that Rho subfamily members are activators of the MAP kinase cascade(28) . Rho proteins (RhoA, RhoB, RhoC) are the specific substrates for C3-like transferases(29, 30, 31, 32, 33, 34) , which inactivate the GTP-binding proteins by ADP-ribosylation at Asn(35) .

The enterotoxin A and the cytotoxin B are the major virulence factors of Clostridium difficile, which is the organism causing the antibiotic-associated diarrhea and pseudomembranous colitis(36) . Recently, it was reported that C. difficile toxin A and B monoglucosylate Rho subfamily proteins(37, 38) . Modification of Rho by these toxins prevents subsequent ADP-ribosylation by C3-like transferases and inhibits the biological activity of the low molecular mass GTP-binding proteins(39, 40) . Because C. difficile toxins are able to enter RBL cells readily, we used toxin B as a novel tool for studying the involvement of low molecular mass GTP-binding proteins of the Rho family in antigen-induced signal transduction. Here we report that toxin B inhibits activation of RBL cells induced by various activators in a manner apparently independent of the actin cytoskeleton.


EXPERIMENTAL PROCEDURES

Materials

C. difficile toxin B(41) , Clostridium botulinum C3 transferase(29) , and C. botulinum C2 toxin (42) were prepared as described recently. 2,4-Dinitrophenyl (DNP)-bovine serum albumin and anti-DNP monoclonal mouse IgE were from Sigma (Deisenhofen, Germany). A polyclonal anti-phosphotyrosine antibody was kindly donated by Dr. G. Schultz (Berlin, Germany). All other reagents were of analytical grade and commercially available. [alpha-P]NAD, [^14C]UDP-glucose, [^3H]serotonin were obtained from NEN Dupont (Bad Homburg, Germany).

Cell Culture

Rat basophilic leukemia (RBL 2H3-hm1) cells (a gift from Dr. G. Schultz, Berlin/Dr. P. Jones, Burlington, VT) were grown in Eagle's minimum essential medium with Earle's salts supplemented with 15% (v/v) heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 6% CO(2) at 37 °C. Cells were incubated overnight with 0.2 µg/ml anti-DNP IgE prior to antigen stimulation experiments. Subclone RBL 2H3-hm1 is transfected with the m1 muscarinic receptor; therefore serotonin release can be induced by carbachol.

Treatment with Toxins

Subconfluent cells were preloaded with anti-DNP-BSA IgE and [^3H]serotonin overnight. Thereafter, the medium was changed and the cells were treated with C. botulinum C2 toxin or C. difficile toxin B for the indicated times and concentrations. From the binary C2 toxin, the concentrations of C2I (enzyme component) are shown in figures and tables; the amount of C2II toxin (binding component) was generally twice as much as indicated for C2I. After toxin treatment cells were washed with the appropriate buffer and used for the assays.

Activation of Cells and Immunoblotting of Tyrosine-phosphorylated Proteins

For tyrosine phosphorylation experiments, cells were seeded in 24-well plates. After cells were nearly confluent, the medium was changed and the cells were incubated with toxin for indicated time and concentrations. Before addition of activating agents, cells were washed three times with RIPA buffer (138 mM NaCl, 6 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 1 mM Na(2)HPO(4), 5 mM NaHCO(3), 5.5 mM glucose, and 20 mM HEPES/NaOH, pH 7.4). The IgE-primed cells were stimulated for 3 min at 37 °C by incubation with DNP-BSA at the indicated concentrations. After addition of 50 µl of modified ice-cold RIPA buffer (150 mM NaCl, 4 mM EDTA, 1 mM Na(3)VO(4), 1% (w/v) desoxycholic acid, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 250 µg/ml p-nitrophenyl phosphate, 20 µg/ml aprotinin, 10 mM Tris/HCl, pH 8.0), cells were mechanically removed, transferred to reaction tubes containing 25 µl of concentrated electrophoresis sample buffer (6% (w/v) SDS, 18% (v/v) 2-mercaptoethanol, 30% (v/v) glycerol, 1 mM Na(3)VO(4), 200 mM Tris/HCl, pH 7.5), and boiled for 8 min. Proteins were subjected to SDS-polyacrylamide gel electrophoresis in 9% gels, followed by transfer of proteins onto nitrocellulose membranes. Phosphotyrosine-containing proteins were detected by incubation with anti-phosphotyrosine antibody (1:1000) for 1 h, the use of horseradish peroxidase-coupled swine anti-rabbit IgG as second antibody, and a chemiluminescence (ECL) Western blotting detection system (Amersham; Braunschweig, Germany).

Serotonin Release

For serotonin release experiments, cells were seeded in 96-well culture plates and incubated overnight with 2 µCi/ml [^3H]serotonin. The cells were then incubated with toxin for the indicated concentrations and time. Thereafter, the medium was removed, and cells were washed three times with RIPA buffer. Incubation at 37 °C with stimuli at the indicated concentrations followed for 25 min. The medium was removed, and the amount of released [^3H]serotonin was quantitated by scintillation counting. Unstimulated cells were treated in the same way as a control for spontaneous release. The values were expressed as percent of the total amount of serotonin. For determination of total amount of serotonin, all cell pellets were lysed in 1% (v/v) Triton X-100 in RIPA buffer, and cell-associated radioactivity was counted.

ADP-ribosylation

ADP-ribosylation was performed as described(29, 43) . The cells were treated with toxin B or C2 toxin (C2I + C2II) and C3 transferase for 2 and 36 h, respectively, at the indicated concentrations. Before cell lysis, the cells were rinsed with ice-cold phosphate-buffered saline and were then scraped off in the presence of lysis buffer (2 mM MgCl(2), 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 25 mM triethanolamine, pH 7.5), sonicated five times for 5 s on ice and centrifuged for 10 min at 1000 times g. The supernatant was used as cell lysate.

The lysates from RBL cells (50 µg) were incubated in buffer (3 mM MgCl(2), 1 mM EDTA, 1 mM dithiothreitol, 50 mM triethanolamine/HCl, pH 7.5) containing 1 µM [alpha-P]NAD (0.5 µCi) and 1 µg/ml ADP-ribosyltransferase C3 for 15 min at 37 °C.

For ADP-ribosylation of actin, cell lysates were incubated in buffer (2 mM MgCl 1 mM dithiothreitol, 10 mM thymidine, 20 mM triethanolamine/HCl, pH 7.5) containing 5 µM [alpha-P]NAD (0.5 µCi) and 1 µg/ml ADP-ribosyltransferase C2I for 15 min at 37 °C. [P]ADP-ribosylated proteins were visualized after 12.5% SDS-PAGE by autoradiography.

Glucosylation

[^14C]UDP glucosylation was performed as described(38) . The cells were treated with toxin B (3 ng/ml) for the indicated time. Before cell lysis, the cells were rinsed with ice-cold phosphate-buffered saline and then scraped off in the presence of lysis buffer, sonicated five times for 5 s on ice, and centrifuged for 1 h at 100,000 times g. The supernatant was used as cytosol. The pellets were resuspended in a buffer containing 50 mM HEPES, pH 7.5, 3 mM MgCl(2), 30 µM GDP. Lysates, cytosols, or membranes (about 50 µg of protein) were incubated with 30 µM [^14C]UDP-glucose and 1 µg/ml toxin B for 1 h at 37 °C. [^14C]UDP-glucosylated proteins were analyzed by 15% SDS-PAGE and by autoradiography (PhosphorImager, Molecular Dynamics).

Two-dimensional Gel Electrophoresis

A combination of isoelectric focusing and SDS-PAGE was used to resolve proteins in two dimensions. First [P]ADP-ribosylated or [^14C]glucosylated proteins were separated in an electric field applying a gradient of pH 5-7, then the probes were run in a 15% SDS-PAGE. Immunoblots with anti-Rho antibody (1:1000) and anti-Cdc42 antibody (1:500) were performed as described above. Modified proteins were visualized by autoradiography.


RESULTS

In RBL cells preloaded with [^3H]serotonin and sensitized with anti-DNP IgE, DNP-BSA stimulated serotonin release in a concentration-dependent manner. A maximal effect was observed at 30 ng/ml. In line with previous reports, serotonin release was reduced at higher concentration of DNP-BSA (Fig. 1A). After treatment with C. difficile toxin B (3 ng/ml), FcRI-mediated degranulation of RBL cells was completely inhibited (Fig. 1A). Toxin B inhibited serotonin release at concentrations as low as 0.3 ng/ml, indicating the specificity of the effect. Maximal inhibition (>90%) was observed at 2 ng/ml after 90 min of incubation (not shown). The time course of the toxin B effect was characterized by a delay of about 30 min, which was most likely due to binding and transfer of the toxin into cells (Fig. 1B). RBL cell activation by carbachol (1 mM) and mastoparan (20 µM), respectively, were also completely blocked by toxin B (3 ng/ml) (Table 1). In contrast, toxin B inhibited serotonin secretion stimulated by the calcium ionophore A23187 maximally by about 50%. Under these conditions, toxin B caused a half-maximal and maximal inhibition at about 0.5 and 2.0 ng/ml, respectively (Fig. 2A). The inhibitory effect of toxin B was largely independent of the concentration of the ionophore (Fig. 2B).


Figure 1: A, effects of C. difficile toxin B on DNP-BSA-induced [^3H]serotonin release. RBL cells were incubated overnight with 2 µCi/ml [^3H]serotonin and primed by DNP-specific IgE. After treatment of cells without (control, bullet) and with toxin B (3 ng/ml, ) for 2 h, medium was removed and cells were washed three times. Degranulation was initiated by addition of DNP-BSA at the indicated concentrations for 25 min at 37 °C. Thereafter, supernatant was removed, and the amount of [^3H]serotonin released was determined by scintillation counting. Data are means ± S.E. of three separate experiments and are given as percent of the total amount of serotonin after correction for spontaneous release (1-3% of total amount). B, time dependence of C. difficile toxin B effects on DNP-BSA-induced [^3H]serotonin release. RBL cells were incubated overnight with 2 µCi/ml [^3H]serotonin and primed by DNP-specific IgE. After treatment without (control, bullet) or with toxin B (3 ng/ml) (toxin B, ) for the indicated times, medium was removed and cells were washed three times. Incubation with 30 ng/ml DNP-BSA followed for 25 min at 37 °C. Thereafter, supernatant was removed and the amount of [^3H]serotonin released was determined by scintillation counting. Data are mean ± S.E. of three separate experiments and are represented as percent of the total amount of serotonin after correction for spontaneous release (1-3% of total amount).






Figure 2: A, concentration dependence of C. difficile toxin B effects on A23187-induced [^3H]serotonin release. RBL cells were incubated overnight with 2 µCi/ml [^3H]serotonin. After treatment without (first point of curve) or with increasing concentrations of toxin B () medium was removed and cells were washed three times. Degranulation was initiated by addition of 300 nM A23187 for 25 min at 37 °C. Thereafter, supernatant was removed, and the amount of [^3H]serotonin released was determined by scintillation counting. Data are means ± S.E. of three separate experiments and are represented as percent of the total amount of serotonin after correction for spontaneous release (1-3% of total amount). B, effects of C. difficile toxin B on A23187-induced [^3H]serotonin release. RBL cells were incubated overnight with 2 µCi/ml [^3H]serotonin. After treatment without (control, bullet) or with toxin B (3 ng/ml) (toxin B, ) for 2 h, medium was removed, and cells were washed three times. Incubation with A23187 at the indicated concentrations followed for 25 min at 37 °C. Thereafter, supernatant was removed, and the amount of [^3H]serotonin released was determined by scintillation counting. Data are means ± S.E. of three separate experiments and are represented as percent of the total amount of serotonin after correction for spontaneous release (1-3% of total amount).



We have shown, recently, that toxin A and B act on the cytoskeleton by inactivation of Rho proteins(39, 40) . Furthermore, we reported that toxin B glucosylates low molecular mass GTP-binding proteins of the Rho subtype family by using UDP-glucose as cosubstrate(40) . Therefore, we studied whether Rho proteins are modified by toxin B also in RBL cells. To this end, lysates from RBL cells, which were pretreated with toxin B (up to 2 h) until the typical morphological changes occurred, were ^14C-glucosylated by toxin B. As shown in Fig. 3A, toxin B induced the incorporation of [^14C]glucose into at least two proteins with M(r) of about 22,000. Pretreatment of intact RBL cells with toxin B reduced subsequent incorporation of [^14C]glucose in the cell lysates, indicating previous modification of proteins in intact cells. We attempted to identify the glucosylated proteins by applying two-dimensional PAGE and immunoblotting. Using anti-RhoA, the upper glucosylated band, which showed a slightly more acidic isoelectric point than the major lower band, was identified as RhoA, whereas the major lower band cross-reacted with anti-Cdc42 antibody (Fig. 3B). A third but minor glucosylated protein with a more acidic isoelectric point than RhoA and an M(r) lower than that of Cdc42 was observed after long exposure. Additionally, RhoA was identified by specific C3-induced ADP-ribosylation. It has been described that glucosylation of Rho at threonine 37 blocks subsequent ADP-ribosylation by C3 at asparagine 41(40) . As shown in Fig. 3C, this was also true for RBL cells. After treatment of intact cells with toxin B, subsequent C3-catalyzed ADP-ribosylation of Rho in cell lysates was inhibited in a toxin B concentration-dependent manner. Thus, Rho was one of the protein substrates of toxin B in intact RBL cells.


Figure 3: A, time dependence of toxin B-induced glucosylation of Rho subfamily proteins in intact RBL cells. RBL cells were treated with toxin B (3 ng/ml) for the indicated times. Thereafter, the medium was removed, cells were washed and scraped off in the presence of lysis buffer. Lysates from control and toxin B-treated cells were incubated with 1 µg/ml toxin B and 30 µM UDP-[^14C]glucose for 1 h at 37 °C. Thereafter, labeled proteins were analyzed by 15% SDS-PAGE. PhosphorImager data from SDS-PAGE are shown. B, identification of toxin B substrates by two-dimensional PAGE and immunoblotting. Lysates from RBL cells were either glucosylated by toxin B or ADP-ribosylated by C. botulinum transferase C3. The lysates were incubated with 1 µg/ml toxin B and 30 µM UDP-[^14C]glucose for 1 h at 37 °C. ADP-ribosylation of the lysates was performed with 1 µg/ml C3 and 0.3 µM [P]NAD (0.5 µCi) for 15 min at 37 °C. After incubation the proteins were separated on two-dimensional gels and electroblotted on nitrocellulose. RhoA was detected with anti-RhoA antibody (b) and Cdc42 with anti-Cdc42 antibody (c). Phosphorimaging of toxin B-catalyzed [^14C]glucosylation is shown in a and C3-catalyzed ADP-ribosylation of Rho in d. Arrows in a indicate three labeled proteins: arrow 1, RhoA; arrow 2, Cdc42, short arrow, not identified. Arrows in b and d indicate RhoA. The arrow in c indicates Cdc42. The asterisk in c indicates a staining artifact that was not observed in repeats. C, time dependence of the effects of C. difficile toxin B in intact RBL cells on subsequent C3-catalyzed ADP-ribosylation of Rho in cell lysates. RBL cells were treated with toxin B (3 ng/ml) for the indicated times. Thereafter, cell lysates were [P]ADP-ribosylated with C3 (1 µg/ml) and 1 µM [P]NAD (0.5 µCi). Labeled proteins were analyzed by SDS-PAGE. PhosphorImager data from SDS-PAGE are shown.



In order to clarify the role of Rho subtype proteins in antigen-induced mast cell activation, we incubated RBL cells with C3 at high concentrations (150 µg/ml) for 36 h and tested the efficacy of the treatment by subsequent [P]ADP-ribosylation of the cell lysate. Although C3 treatment of intact cells caused significant reduction in labeling of Rho in the cell lysates (Fig. 4), antigen-induced mast cell activation was not affected by C3 (Table 1).


Figure 4: ADP-ribosylation of Rho in intact RBL cells. RBL cells were treated for 36 h with the indicated concentrations of C3. Thereafter, ADP-ribosylation of the lysates was performed with 1 µg/ml C3 and 1 µM [P]NAD (0.5 µCi) for 15 min at 37 °C, and labeled proteins were analyzed by SDS-PAGE. PhosphorImager data from SDS-PAGE are shown.



It has been suggested that Rho subfamily proteins are involved in the regulation of tyrosine phosphorylation(44) . Therefore, we studied the effects of toxin B on FcRI-mediated tyrosine phosphorylation by detecting protein tyrosine phosphorylation with anti-phosphotyrosine antibody. Antigen-receptor activation by DNP-BSA largely increased tyrosine phosphorylation of various proteins (not shown). Fig. 5A shows that toxin B selectively blocked tyrosine phosphorylation of a protein of about 110 kDa. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, which was shown to block antigen-induced mast cell activation (45) (Table 2), similarly inhibited protein tyrosine phosphorylation of the 110-kDa protein (Fig. 5B). However, in A23187-activated RBL cells, tyrosine phosphorylation of several proteins were inhibited by toxin B, but not by wortmannin. Moreover, toxin B and wortmannin inhibited degranulation of RBL cells in an additive manner (Table 2), indicating that both agents differ in their modes of action.


Figure 5: A, effects of C. difficile toxin B on tyrosine phosphorylation in RBL cells. Cells were primed overnight with anti-DNP IgE. After incubation without(-) or with toxin B [3 ng/ml] (+) for 2 h, cells were washed and stimulated for 3 min at 37 °C with DNP-BSA (500 ng/ml) or A23187 (0.5 µM). Thereafter, cells were lysed and proteins analyzed by SDS-PAGE. Immunoblotting was performed with anti-phosphotyrosine antibody and detected with the ECL Western blotting detection system (Amersham). Chemiluminogram is shown. Arrows indicate tyrosine phosphorylation of proteins which is inhibited after incubation with toxin B. B, effects of wortmannin (wort.) on tyrosine phosphorylation in RBL cells. Cells were incubated overnight with anti-DNP IgE. After incubation without (control (con.), 2 h) and with toxin B [3 ng/ml] (toxin B, 2 h) or wortmannin [100 nM, 15 min] cells were washed and stimulated for 3 min at 37 °C with DNP-BSA [500 ng/ml] or A23187 [0.5 µM]. Thereafter cells were lysed and proteins analyzed by SDS-PAGE. Immunoblotting was performed with anti-phosphotyrosine antibody and detection with the ECL Western blotting detection system (Amersham). Chemiluminogram of DNP-BSA-stimulated protein tyrosine phosphorylation is shown in I and of A23187-induced protein tyrosine phosphorylation in II. Arrows indicate inhibition of tyrosine phosphorylation of proteins by toxin B.





C. difficile toxin B is a well known cytotoxin(46) . Its cytotoxic effects are characterized by rounding up of cells and disassembly of the actin cytoskeleton(47) . Because cytoskeleton rearrangement is thought to be involved in FcRI signal transduction (48, 49) , it was of interest to clarify whether redistribution of the actin cytoskeleton is the mechanism by which toxin B inhibits antigen-induced serotonin release. Therefore, the influence of C2 toxin on FcRI-mediated RBL cell activation was tested. C2 toxin ADP-ribosylates actin thereby inhibiting actin polymerization and causing depolymerization of F-actin(50, 51, 52, 53) . As shown in Fig. 6A, treatment of RBL cells with C2 toxin, which similarly induced rounding up of cells (not shown), substantially increased the stimulated release of serotonin from RBL cells. This effect was observed with antigen as well as with ionophore-stimulated exocytosis, but was not caused by alteration of the basal serotonin secretion. C2 toxin-induced [P]ADP-ribosylation of actin was largely reduced in lysates from RBL cells pretreated for 2 h with C2 toxin, indicating that cellular actin was ADP-ribosylated in intact cells (Fig. 6B). A comparable increase in serotonin release occurred in the presence of cytochalasin D (5 µM; not shown). However, even in the presence of C2 toxin, C. difficile toxin B inhibited antigen- and ionophore-mediated serotonin release (Table 1). This effect was also observed when RBL cells were pretreated with C2 toxin for 1 h (not shown). Thus, all these findings suggest that microfilament depolymerization is not causally involved in toxin B-induced inhibition of antigen-stimulated serotonin release.


Figure 6: A, effects of C. botulinum C2 toxin on DNP-BSA-induced [^3H]serotonin release. Cultures were incubated overnight with 2 µCi/ml [^3H]serotonin and primed by anti-DNP-IgE. After treatment without (control, bullet) or with C2 toxin (100 ng/ml C2I + 200 ng/ml C2II) (C2 toxin, ) for 2 h, medium was removed, and cells were washed three times. Incubation with DNP-BSA at the indicated concentrations followed for 25 min at 37 °C. Thereafter, supernatant was removed, and the amount of [^3H]serotonin released was determined by scintillation counting. Data are means ± S.E. of three separate experiments and are represented as percent of the total amount of serotonin after correction for spontaneous release (1-3% of total amount). B, time dependence of ADP-ribosylation of actin by C. botulinum C2 toxin. RBL cells were incubated without or with C2 toxin (100 ng/ml C2I + 200 ng/ml C2II) for the indicated times. Thereafter, the medium was removed, and cells were scraped off in the presence of lysis buffer. Lysates from control and C2 toxin-treated cells were incubated with 1 µg/ml C2I and 5 µM [P]NAD for 15 min at 30 °C. After incubation the proteins were separated by 12.5% SDS-PAGE. PhosphorImager data from SDS-PAGE are shown. Incubation with enzyme component (C2I) or binding component (C2II) alone was performed as controls.




DISCUSSION

Here we report that the cytotoxin C. difficile toxin B inhibits antigen-stimulated serotonin release from RBL 2H3-hm1 cells. Because C. difficile toxins A and B have been shown to act on Rho subfamily proteins(37, 38) , we conclude that inhibition of IgE-stimulated serotonin release is caused by the action of the toxin B on this family of GTP-binding proteins and suggest that Rho subfamily proteins are involved in the FcRI signal pathway. Involvement of Rho proteins in the action of C. difficile toxins was first deduced from the finding that the toxins inhibit C3-induced ADP-ribosylation of Rho proteins(39, 40) . Recently, the modification of Rho subfamily proteins by C. difficile toxin was identified as monoglucosylation at threonine 37 of Rho. Here we show that C. difficile toxin B catalyzes the glucosylation of Rho proteins in intact RBL cells. As deduced from the inhibitory effect of toxin B on C3-induced ADP-ribosylation, which is selective for RhoA, RhoB, RhoC, and from immunoblotting with anti-RhoA antibody, we conclude that RhoA is one protein substrate for C. difficile toxin in RBL cells. In addition, glucosylation and immunoblot studies indicate that also Cdc42 is modified by toxin B. Additionally, a minor glucosylated protein was detected after extended phosphorimaging of two-dimensional PAGE. So far, the nature of this protein is not clear. It was shown that recombinant Rac is also substrate for glucosylation by toxin B from C. difficile(38) . However, applying two-dimensional PAGE and immunoblotting with anti-Rac antibody, we did not detect glucosylated Rac in RBL cell lysate.

Various GTP-binding proteins have been postulated to be involved in exocytic processes from mast cells. A pertussis toxin-sensitive G-protein is involved in mast cell activation by compound 48/80 but not in FcRI-mediated signal transduction(15, 16) . Studies with permeabilized mast cells led to the hypothesis that a GTP-binding protein, G(E), which is part of the membrane fusion mechanism, is involved in exocytosis(13, 14, 54) . However, whether G(E) is actually a heterotrimeric G-protein or not remains to be clarified. Furthermore, recent studies indicated that activation of mast cells by compound 48/80 is paralleled by redistribution of the actin filaments, a phenomenon which may involve Rho and Rac proteins besides a heterotrimeric G-protein(55) . Moreover, Price et al.(56) reported that the GTP-binding proteins Rac and Rho are involved in GTPS-induced secretion from permeabilized mast cells. Our study with C. difficile toxins also suggests that Rho subfamily proteins participate in RBL cell activation. However, treatment of RBL cells with C3 at high concentrations, which resulted in ADP-ribosylation of at least 90% of Rho in intact cells, did not inhibit antigen-induced secretion from RBL cells. Therefore, we conclude that glucosylation of Cdc42 rather than Rho is responsible for inhibition of antigen-stimulated secretion in these cells. Because Rho subfamily proteins (including Cdc42) are involved in regulation of the actin cytoskeleton(17) , the question arises whether the perturbation of the actin cytoskeleton by toxin B is responsible for inhibition of antigen-stimulated secretion. We addressed this question by using C. botulinum C2 toxin and cytochalasin D, which directly act on actin. C2 toxin ADP-ribosylates G-actin, thereby inhibiting actin polymerization and causing depolymerization of microfilaments(50, 51) . Cytochalasin D acts like a capping molecule on F-actin to inhibit actin polymerization(57) . Because depolymerization of actin by C2 toxin or cytochalasin increased rather than decreased antigen- and ionophore-stimulated secretion, the regulatory functions of Rho subfamily proteins on the actin cytoskeleton are not causal for the C. difficile toxin-induced inhibition of RBL cell activation. On the contrary, the data indicate that these small GTP-binding proteins play a central role in early signal transduction processes, i.e. upstream of regulation of the actin cytoskeleton or vesicle fusion processes. This is supported by the findings that antigen-, carbachol-, and mastoparan-induced serotonin release were completely inhibited by toxin B, whereas ionophore-induced release was only partially affected. Rho subfamily proteins regulate various enzyme systems involved in signal transduction for example phosphatidylinositol-4-phosphate-5-kinase(25) , phospholipase D(24) , or phosphatidylinositol 3-kinase(26) . Cdc42, which appears to be the major substrate for toxin B-induced glucosylation in RBL cells, was shown to interact with p85, the regulatory component of phosphatidylinositol 3-kinase thereby increasing kinase activity(58) . Therefore, we compared the action of toxin B with wortmannin, which inhibits the catalytic subunit of phosphatidylinositol 3-kinase (p110) (45) . As shown earlier(45) , wortmannin potently blocked the antigen-stimulated RBL cell secretion. However, at least two findings indicate that toxin B and wortmannin might act on different signal pathways. First, tyrosine phosphorylation pattern of ionophore-activated RBL cells were different with both agents. Second, toxin B and wortmannin inhibited activation of RBL cells in an additive manner. Thus, the target GTPases of toxin B are not only involved in phosphorylation reactions within the signal pathway of phosphatidylinositol 3-kinase but may also regulate additional signal pathways. However, at present it is not clear whether the decrease in tyrosine phosphorylation observed after toxin B treatment is due to a direct or indirect effect.

Taken together, we employed C. difficile toxin B as a novel tool for studying the involvement of low molecular mass GTP-binding proteins of the Rho subfamily in FcRI-mediated cell activation. Complete inhibition of antigen-induced activation indicates an essential role for these regulatory GTPases in RBL cell activation. As Cdc42 and Rho are the major targets for glucosylation by toxin B and Rho inactivation by C3 was without effect on secretion, we suggest that Cdc42 is basically involved in signal transduction via the FcRI receptor in RBL cells.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246 project B10, DFG Ju 231/3-1 and Ei 206/3-1) and by the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Tel.: 49-761-2035301; Fax: 49-761-2035311.

(^1)
The abbreviations used are: RBL, rat basophilic leukemia; RBL 2H3-hm1, 2H3-hm1 subline of rat basophilic leukemia cells; A23187, calcium ionophore A23187; C2 toxin, C. botulinum C2 toxin; C2I, enzyme component of C. botulinum C2 toxin; C2II, binding component of C. botulinum C2 toxin; C3, ADP-ribosyltransferase from C. botulinum; DNP-BSA, dinitrophenyl-conjugated bovine serum albumin; FcRI, high affinity receptor for IgE; G-protein, GTP-binding protein; IgE, immunoglobulin E; PAGE, polyacrylamide gel electrophoresis; toxin B, C. difficile toxin B; GTPS, guanosine 5`-O-(thiotriphosphate).


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