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TOXICOLOGY
Section of Molecular Biology, Department of Biology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland
Received January 9, 2004; accepted May 6, 2004.
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Abstract |
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). Synaptic transmission involves both vesicle trafficking and rearrangement of the actin cytoskeleton, processes that are directed by members of the Ras family of low molecular weight GTPases, namely, Rho, Rac, and Cdc42 (Postma et al., 1996; Jaffe and Hall, 2002). These GTPases are isoprenylated at their C terminus (Huang et al., 2001) and cycle between GDP-bound (inactive) and GTP-bound (active) states that are regulated primarily by three classes of molecules: guanine nucleotide exchange factors, GTPase-activating proteins, and GDIs (Van Aelst and D'Souza-Schorey, 1997). Thus, Rho, Rac, and Cdc42 serve as molecular switches that regulate vesicle trafficking and cytoskeletal dynamics during exocytosis.
The phospholipid lysophosphatidic acid (LPA) elicits a variety of biological effects in many cell types by evoking rapid and transient Ca2+ signaling (Morgan, 1995; Moolenaar, 1999) through its heterotrimeric G protein-coupled receptor (Gohla et al., 1998). LPA also promotes actin cytoskeletal changes through a Rho-mediated signaling pathway (Postma et al., 1996). This actin reorganization in neuronal cells is considered to be prerequisite to neurotransmitter release from presynaptic neurons (Trifaro et al., 1993). In PC12 cells, Shiono et al. (1993) showed that LPA stimulates dopamine release mediated by LPA receptors. Komuro et al. (1996) and Mariot et al. (1996) found that RhoGDI inhibits exocytosis in nerve growth factor (NGF)-treated PC12 and mast cells, presumably by preventing GTP/GDP exchange as well as GTP hydrolysis. These data suggest that LPA-stimulated Rho signaling plays an important role in exocytosis in PC12 cells.
Recent work suggests that Ca2+-induced exocytosis of the neurotransmitter acetylcholine (ACh) is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein target receptors that were originally identified as factors required for synaptic vesicle fusion (Blasi et al., 1993). Soluble N- ethylmaleimide-sensitive factor attachment protein target receptors are specific substrates for proteolysis by botulinum toxins (Keller et al., 1999; Lawrence and Dolly, 2002). Botulinum toxins are clostridial neurotoxins that comprise seven different serotypes, designated A to G. Botulinum toxin type A (BoNT/A) inhibits ACh release at peripheral cholinergic synapses by the metalloprotease activity of its light chain (50 kDa) on 25-kDa synaptosomal-associated membrane protein (SNAP-25), a protein that is essential for membrane fusion. In this manner, BoNT/A causes peripheral neuromuscular paralysis, making it one of the most potent neurotoxins known to mankind (Blasi et al., 1993). However, deletion of SNAP-25 does not result in a complete blockade of exocytosis in NGF-treated PC12 cells (Ray et al., 1997). Furthermore, Ca2+ influx and cAMP elevation overcomes BoNT/A-mediated inhibition of insulin secretion (Huang et al., 2001). These findings suggest that there may be another pathway through which BoNT/A inhibits exocytosis.
In this study, we focused on the role of Rho GTPases and the actin cytoskeleton on regulated ACh exocytosis in NGF-treated PC12 cells. We have demonstrated that LPA stimulates Ca2+-dependent ACh release via actin reorganization through a RhoB-mediated pathway and that BoNT/A blocks this release by affecting the levels of RhoB, thereby stabilizing actin and inhibiting its reorganization. We show that overexpression of wild-type RhoB (wRhoB) overcomes the inhibitory effect of botulinum toxin on actin reorganization as well as ACh release stimulated by KCl or LPA. On the contrary, overexpression of a dominant negative RhoB causes inhibition of ACh release and actin reorganization by either KCl or LPA regardless of the toxin treatment.
Finally, we show that knockdown of RhoB gene expression by small interfering RNA (siRNA) results in a total blockade of ACh release and actin reorganization stimulated by LPA. We conclude that RhoB plays an important role in controlling neurotransmitter release by regulating actin cytoskeletal reorganization in PC12 cells and that BoNT/A affects this pathway.
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Materials and Methods |
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Cell Culture. PC12 cells were obtained from BD Biosciences Clontech. Cells were seeded at a density of 4 x 104 cells/cm2 in Dulbecco's modified Eagle's medium containing 7% fetal calf serum and 10% donor horse serum, streptomycin (100 µg/ml), and penicillin (100 U/ml). NGF at a final concentration of 50 ng/ml was added to the culture medium 24 h after seeding, and cells were maintained for 4 to 5 days during which the medium was changed every other day (to achieve NGF-treated cells) at 37°C in a CO2 incubator. Cells grown to 80% confluence were exposed to 10 nM BoNT/A for 4 to 24 h as described in the figure legends.
ACh Release Study. ACh release was measured according to the method of Ray et al. (1993). Briefly, NGF-treated monolayer cells at about 90% confluence were prelabeled with 10 µM [methyl-3H]choline chloride at 37°C for 1 h in HEPES-buffered solution (medium A: 116 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 0.81 mM MgCl2, and 25 mM HEPES adjusted to pH 7.4 and to 340 mOsM/l with NaCl). The cells were washed to remove the unincorporated radiolabeled compound and were then detached by gentle shaking and centrifuged at 600g to for 2 min to obtain a pellet that was suspended in 1 ml of isosmotic medium A with 20 µM eserine sulfate (medium B). Radiolabeled cells were washed inside a custom-designed perfusion chamber as described previously (Ray et al., 1993) for 8 min with medium B, and then perfused for 8 min with medium B containing ACh release stimulant (either 80 mM KCl or 0.2–100 µM LPA) followed by another 8-min wash in medium B. The amount of [3H]ACh in each fraction was assayed by the choline kinase method (Ray et al., 1993). The protein content of perfused cells was determined using the bicinchoninic acid protein assay kit as per the manufacturer's instructions, and the results are expressed as femtomoles of ACh released per milligram of protein per minute.
Actin Localization and Reorganization. Actin architecture was visualized by immunohistochemistry as described previously (Trifaro et al., 1993), with some modifications. Briefly, PC12 cells grown on plastic slides for 3 days with NGF and then the medium was replaced with medium A for 1 h followed by addition of LPA (final concentration 10 µM). The cells were incubated further for 5 to 10 min. Then, the cells were rinsed twice with PBS and fixed with 3.7% (w/v) paraformaldehyde for 10 min at 37°C. After rinsing with PBS followed by blocking with PBS containing 1% bovine serum albumin, cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature followed by actin staining by incubation with phalloidin-tetramethylrhodamine isothiocyanate at 4°C for 1 h. Specimens were mounted with the anti-fade reagent ProLong and maintained overnight at 4°C. Immunocomplexes (actin filaments) were examined by Bio-Rad confocal and/or Nikon fluorescence microscopy imaging systems. For the confocal microscopic images, the focal plane was set at the middle of the cells.
Western Blotting to Quantitate the Levels of RhoB, RhoA, or NSE Expression. To analyze the expression levels of RhoA, RhoB, and NSE, cells grown on plastic six-well tissue culture plates were solubilized in 200 µl of ice-cold lysis buffer [M-PER containing 0.1 mM EDTA, 0.1 mM EGTA, and 5% (v/v) proteinase inhibitor cocktail] in a 1.5-ml tube. After centrifugation at 12,000 rpm for 30 min at 4°C, the supernatant were transferred into a new 1.5-ml tube and boiled in SDS-polyacrylamide gel electrophoresis sample buffer [0.9 M Tris-HCl, pH 8.45, 24% (w/v) glycerol, 4% SDS, 0.015% bromphenol blue G, and 0.005% phenol red] for 3 min. Then, the samples were loaded onto 12% Tris/glycine precast gradient polyacrylamide gels and were separated by SDS-polyacrylamide gel electrophoresis for Western blotting. After electrophoresis, the protein bands on the gel were transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in PBS for 1 h followed by incubation with first antibodies described in the figure legends. After washing, the membranes were further incubated with secondary antibody conjugated with horseradish peroxidase [diluted 1:3000 in PBS containing 0.05% (w/v) Tween 20].
Inhibition of RhoB Degradation by Proteasome Inhibitors. Stock solutions of proteasome-specific inhibitors (lactocystin, MG-132, and proteasome inhibitor I) were dissolved in 100% dimethyl sulfoxide at a concentration of 10 mM each. At the end of culture, the growth medium of PC12 cells was replaced with medium A containing PIs (each at 10 µM final concentration, with dimethyl sulfoxide at 0.3% final concentration) and incubated at 37°C for 2 h followed by stimulation with LPA for 10 min. Then, the cells were solubilized for subsequent Western blotting analysis as described above.
Expression Constructs and Overexpression of wRhoB and a Mutant RhoB. The rat wRhoB and a truncation mutant of RhoB (tRhoB) lacking the C-terminal peptide CKVL, the active motif for posttranslational modification for RhoB to function (Sebti and Hamilton, 2000; Prendergast and Rane, 2001) containing EcoR1 and XbaI restriction sites for cloning, were amplified by polymerase PCR from rat brain cDNA. The primers for wRhoB and tRhoB were 5'-ATGGCGGCCATCCGCAAGAAG-3' and 5'-TGACGACGTTCCACGATACT-3', and 5'-ATGGCGGCCATCCGCAAGAAG-3' and 5'-TCAGCAGTTGATGCAGCCGTTC-3', respectively. PCR was performed in a final volume of 25 µl containing PCR beads, 100 pmol of each primer, and 200 ng of template rat cDNA. Reactions were subjected to 20 cycles of denaturation at 94°C for 2 min, annealing at 56°C for 1 min, and elongation at 72°C for 1 min. PCR products were then cloned into pTracerCMV by using standard techniques. Constructs were sequenced to confirm the right frame (data not shown). For overexpression of wRhoB or tRhoB, NGF-treated PC12 cells were transiently transfected using LipofectAMINE 2000 according to the manufacturer's protocol, using 1 to 10 µg of each construct. Overexpression was confirmed 48 h after transfection by Western blotting (data not shown).
RNA Interference. QIAGEN-Xeragon (Germantown, MA) chemically synthesized the sense and antisense strands for two 21-nucleotide regions of the rat RhoB gene (5'-CAAUGUGCCCAUCAUCUUGGU-3' and 5'-AAAAAAGACCUGCGCAGCGAU-3'), 252 and 324 nucleotides downstream of the start codon, respectively. As a control siRNA duplex, the luciferase GL2 siRNA, whose targeting sequence is 5'-CGUACGCGGAAUACUUCGA-3', was obtained from Dharmacon (Lafayette, CO). Twenty-four hours before transfection of each siRNA duplex, PC12 cells were plated in growth medium containing NGF at a density of 2 x 106 cells/dish in 10-cm culture dishes (for both ACh and Western blotting studies) or 4.0 x 104 cells in a plastic chamber slide (for microscopy). Transfection was carried out according to the manufacturer's protocol using Oligofectamine (Invitrogen). Twenty-four hours after transfection, the cells were subjected to a ACh release study, Western blotting analysis, RNA extraction followed by RT-PCR analysis, and actin staining as described above.
RT-PCR Analysis. The same primers for PCR cloning of RhoB were used for RT-PCR analysis to estimate RhoB mRNA levels with or without siRNA duplex transfection. Total RNA was extracted by using TRIzol reagent (Invitrogen) and used as a template for RT-PCR. Reverse transcription and amplification of total RNA (100 ng) was accomplished according to the manufacturer's protocol in a one-step format kit (BD Biosciences Clontech). The PCR products were analyzed by agarose gel electrophoresis, and the images were captured by Gel Doc 2000 (Bio-Rad).
Statistical Analysis. The number of replicated experiments is noted in each figure legend. Values represent means ± S.E.M. of experimental replicates. Significant differences were determined using the t test within the Sigma Plot program (SPSS Inc., Chicago, IL). Statistical significance was set at p < 0.05.
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1.5- to 2.5-fold over the basal release rate (no LPA; Fig. 1A). Figure 1B shows typical changes in ACh release over time after stimulation by KCl (80 mM) or LPA (10 µM). After addition of either LPA or KCl, ACh release increased by severalfold over the basal level and subsequently declined and plateaued in each case. The amount of ACh release by LPA (10–100 µM) was equivalent to levels triggered by high K+ (80 mM) known to trigger depolarization- and Ca2+-induced exocytosis (Fig. 1A).
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Actin Reorganization by LPA and the Stabilization of Actin Architecture by BoNT/A. Actin reorganization is a prerequisite for exocytosis (Trifaro et al., 1993). Therefore, the effect of LPA on actin architecture in NGF-treated PC12 cells was investigated before and after treatment with 10 µM LPA for 10 min and/or 10 nM BoNT/A for 24 h using both fluorescence microscopy (FM) and confocal microscopy (CM). In untreated cells, intensely stained actin rings were visualized along the outer limiting cell membranes, whereas very diffuse actin staining was apparent in the cytosol (Fig. 2, A and E). In LPA-stimulated (10 µM; 10 min) cells, FM revealed the absence of the ring but showed actin structures throughout the cytosol (Fig. 2B). CM confirmed that actin reorganization resulted in the loss of the actin ring and the accumulation of filamentous actin structures throughout the cytosol within 10 min after the stimulation (Fig. 2F). BoNT/A treatment alone did not promote actin organization (Fig. 2, C and G). When cells were pretreated with 10 nM BoNT/A for 24 h followed by exposure to LPA (10 µM) for 10 min, no such actin reorganization was observed along the membrane or in the cytosol (Fig. 2, D and H), suggesting that BoNT/A stabilized the actin architecture. Although the data were not shown in the figure, the same inhibition of actin reorganization was observed with KCl stimulation.
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RhoB Associated with LPA and/or BoNT/A Treatment. Rhos are key regulators of the actin cytoskeleton in various cells (Takai et al., 2001). However, it is not known which Rho(s) regulates actin filament assembly involved in PC12 cell exocytosis. Because RhoB is inducible as well as degradable (Engel et al., 1998), we tested whether treatment with BoNT/A and/or LPA affects the level of RhoB using Western blotting. RhoB levels were not altered when cells were treated with either 10 µM LPA for 10 min or BoNT/A (1–100 nM) for 24 h (Fig. 3A). However, a marked dose-dependent decrease in RhoB immunoreactivity was evident in cells treated with BoNT/A followed by LPA stimulation for 10 min (Fig. 3A). No such difference in immunoreactivity was evident for NSE (internal control), suggesting that the decrease in RhoB was not due to a general decrease in protein levels (Fig. 3A). As shown in Fig. 3B, a 24-h treatment of NGF-treated PC12 cells with BoNT/A caused a marked decrease in SNAP-25 (Fig. 3B), indicating successful translocation of the light chain of BoNT/A into NGF-treated PC12 cells. The exact mechanism by which LPA plus BoNT/A cause RhoB degradation remains to be established.
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Effects of wRhoB or tRhoB Overexpression on the Inhibition of ACh Release in the Presence or Absence of BoNT/A. PC12 transfectants that overexpressed wRhoB released about 4-fold more ACh upon LPA stimulation compared with normal PC12 cells (Fig. 4A). However, overexpression of tRhoB did not cause ACh release in response to LPA, suggesting that this truncation mutant inhibited ACh release. There was no difference in the basal (no LPA) levels of ACh release among the normal cells and transfectants with either wRhoB or tRhoB (Fig. 4A). PC12 cells transfected with vector alone showed the same amount of LPA-induced ACh release as untransfected cells (2-fold over basal level), indicating that vector transfection had no effect on either basal or stimulated ACh release (Fig. 4A). Interestingly, when BoNT/A was tested, PC12 transfectants that overexpressed wRhoB did not show the expected decrease in ACh release after LPA or KCl treatment, suggesting that wRhoB overexpression antagonized the toxin's inhibitory effect on exocytosis (Fig. 4A). Figure 4B shows a representative time course of ACh release in the transfectants with or without BoNT/A treatment. There was no difference among the time courses for the control cells, the transfectants, and the transfectants treated with BoNT/A. Western blotting confirmed that both RhoB constructs were expressed at a high level 24 h after transfection (data not shown). The transfection efficiency was 30 to 35%, as estimated by FM using excitation and emission wavelengths of 395 and 507 nm, respectively (data not shown).
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Effects of wRhoB or tRhoB Overexpression on Actin Reorganization by LPA and Stabilization of Actin Architecture by BoNT/A. Figure 5 shows the effects of LPA and/or BoNT/A treatment on actin architecture in wRhoB- or tRhoB-overexpressing PC12 cells. Cells transfected with vector alone (Fig. 5E) showed no change in the basic actin architecture compared with control cells (no treatment; Fig. 5A), in that strong actin staining was evident as a ring along the cell membrane. Interestingly, wRhoB overexpression alone resulted in the reorganization of actin without LPA (10 µM) stimulation because the ring disappeared, whereas the cytosol accumulated actin filaments (Fig. 5B). BoNT/A treatment for 24 h did not interfere with this actin reorganization in the wRhoB-overexpressing transfectants regardless of LPA treatment (Fig. 5, C and D). The actin ring was maintained after transfection of cells with the mutant tRhoB, and there was no accumulation of cytosolic actin (Fig. 5, F–H) with BoNT/A and/or LPA treatment. These data indicate that BoNT/A inhibits LPA-stimulated actin reorganization and that overexpression of wRhoB promotes actin reorganization and overcomes the toxin's inhibitory effect on actin reorganization by LPA. The data also strongly suggest that the RhoB signaling pathway plays an important role not only in actin reorganization but also in exocytosis stimulated by LPA (via regulating actin reorganization). Furthermore, BoNT/A may target RhoB thus interfering with ACh release by blocking LPA-stimulated actin rearrangement in NGF-treated PC12 cells.
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RhoB Degradation by Proteasomes. Because proteasomes degrade many short-lived regulatory proteins involved in the cell cycle or apoptosis, we also investigated whether the decrease in RhoB levels was due to degradation by this pathway (Ciechanover, 2001). Therefore, we used combinations of the specific proteasome inhibitors lactacystin, MG132, and proteasome inhibitor I to study whether the rapid decrease in RhoB associated with BoNT/A and LPA treatment was due to degradation by 26S proteasomes. When BoNT/A-treated (10 nM for 24 h) cells were incubated for 2 h in medium A containing proteasome inhibitors and then stimulated with LPA, RhoB immunoreactivity returned to control levels (Fig. 6A), suggesting that the decrease in RhoB was due to proteasome-mediated degradation. In contrast, no change in RhoA immunoreactivity was evident in the control (no treatment) or in LPA- and/or BoNT/A-treated cells either in the presence or absence of PIs (Fig. 6B). These data suggest that RhoB, but not RhoA, is degraded by proteasomes.
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RNA Interference. To establish the role that RhoB plays in ACh release and actin reorganization, we used siRNA to deplete NGF-treated PC12 cells of endogenous RhoB. The cells were transfected with two distinct siRNAs that targeted two different wRhoB mRNA sequences. Western blotting and RT-PCR revealed a marked reduction in RhoB protein (Fig. 7A) and a complete knockdown of the mRNA (Fig. 7B), respectively, at 24 h post-transfection with two siRNA duplexes. Single transfection with either targeted siRNA did not reduce the level of RhoB or its mRNA (data not shown), and neither the protein nor the mRNA was reduced when cells were transfected with an siRNA duplex specific for luciferase, relative to the control (no transfection; data not shown).
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The effect of RhoB knockdown on ACh release and actin reorganization stimulated by LPA was studied at 24 h post-transfection of the two targeted siRNA constructs. Transfection with RhoB-specific siRNAs inhibited ACh release by LPA, whereas transfection with the luciferase duplex had no effect (Fig. 8A). Similarly, LPA-induced actin reorganization also was blocked by transfection with RhoB-specific siRNAs, because brightly stained actin rings were evident along the cell membrane regardless of LPA treatment (Fig. 8B, c and d). However, LPA-induced actin reorganization was not affected in cells transfected with the luciferase siRNA (Fig. 8B, b). Given that exocytosis in all cells is dependent on actin reorganization, these results suggest that RhoB plays a critical role in neuroexocytosis in PC12 cells.
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Discussion |
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). LPA is a pleiotropic phospholipid growth factor that activates the same signal transduction mechanism that is activated by polypeptide growth factors via specific G protein-coupled receptors that subsequently activate small GTPases (Moolenaar, 1999). Our data from NGF-treated PC12 cells indicate that LPA-stimulated ACh release is controlled by G protein-related signal transduction as well. Significantly, it is reported that Rac1, a member of the Rho family, regulates neurotransmitter release in Aplysia neurons (Doussau et al., 2000). Although data are not shown, preincubation of cells in medium A containing both 1 mM EGTA and 20 µM BAPTA for 30 min inhibited ACh release stimulated by either KCl (80 mM) or LPA (10 µM), suggesting that LPA stimulates Ca2+-dependent neuroexocytosis in NGF-treated PC12 cells.
Because exocytosis is dependent on cytoskeletal components (Trifaro et al., 1993), we show that LPA stimulates ACh release in NGF-treated PC12 cells by inducing actin reorganization, a prerequisite for exocytosis. As shown in Fig. 2, intense actin staining in rings and sparse distribution in the cytosol was observed in untreated control cells under both FM and CM. Microscopy revealed the rapid disappearance of the ring and redistribution of actin in the cell body after the addition of LPA to the culture medium. This indicates that LPA induces the reorganization of the actin cytoskeleton. Similar actin reorganization was observed when cells were treated with KCl (data not show). Similarly, disappearance of the actin ring and actin redistribution was reported after treatment of cultured primary neurons with 40 mM KCl (Soltysik-Espanola et al., 1999). We found that BoNT/A alone did not alter the actin architecture, but rather inhibited actin reorganization induced by LPA, suggesting that the toxin stabilizes actin dynamics (either directly or indirectly).
Although all Rho family members are thought to regulate actin reorganization to some extent (Van Aelst and D'Souza-Schorey, 1997), it is not clear which Rho protein is responsible for LPA-induced actin dynamics in PC12 cells. Rho proteins exhibit diverse physiological functions, and elevated levels have been associated with cancer (Prendergast, 2001). RhoB is distinct among the Rho family with respect to the following facts: 1) only RhoB is both inducible and degradable; and 2) RhoB has a short half-life, suggesting that its function in signaling processes is dependent on its concentration as well as the GTP/GDP switch (Engel et al., 1998). Accordingly, we followed RhoB expression in cells after exposure to BoNT/A and/or LPA stimulation. Only RhoB degradation occurred in cells cotreated with BoNT/A and LPA (Fig. 3A). No such degradation was observed with RhoA (Fig. 6B), which has the highest sequence similarity to RhoB at the DNA level. The fact that the exposure of NGF-treated PC12 cells to BoNT/A for 24 h resulted in degradation of both SNAP-25 (Fig. 3B) and RhoB (Fig. 3A) in this experimental model may suggest that RhoB is another target for BoNT/A.
To test whether RhoB plays a crucial role in actin reorganization leading to ACh release in NGF-treated PC12 cells, mammalian expression constructs encoding either full-length wRhoB or a four-amino acid C-terminal truncation mutant of RhoB (tRhoB) were generated by PCR cloning. Overexpression of tRhoB resulted in the stabilization of the actin rings to inhibit both actin reorganization and ACh release by LPA (Figs. 4, and 5). In contrast, wRhoB overexpression caused actin reorganization in the absence of LPA stimulation and enhanced ACh release upon LPA stimulation. These findings strongly suggest that actin reorganization is requisite for ACh release and that RhoB is involved in regulating this step, perhaps through changes in its expression level. Because RhoA, B, and C control actin reorganization in various cells through common Rho downstream effectors, including Rho-associated kinase (Maekawa et al., 1999), RhoB overexpression most likely resulted in activation of these effectors.
The rapid decrease in RhoB levels prompted us to further examine the possibility that it is degraded via the proteasome pathway, as are many short-lived proteins such as p53, cyclins, and I
B (Rolfe et al., 1997; Adams et al., 1999). These proteins undergo rapid proteasomal degradation, suggesting that timed destruction of cellular regulators by the proteasomes is critical for regulating various cellular processes (Tsubuki et al., 1993). As Fig. 6A demonstrates, proteasome inhibitors halted the decrease of RhoB in the presence of BoNT/A and LPA, strongly suggesting that RhoB is degraded by proteasomes. In contrast, under identical conditions RhoA levels were unchanged (Fig. 6B), suggesting that RhoB, but not RhoA, is responsible for actin rearrangement and ACh release in response to LPA.
Giry et al. (1995) showed that overexpression of Rhos protects cells from Clostridium difficile toxin B, presumably by inhibiting this toxin's enzymatic activity. Spyres et al. (2003) also reported that the nonenzymatic portion of C. difficile toxin B inhibits its toxicity. These reports support our results that BoNT/A targets RhoB, a finding that may facilitate the development of new drugs to counter this toxin and thereby lessen the virulence of Botulinum in humans. However, the mechanism by which the BoNT/A light chain triggers RhoB degradation upon LPA stimulation remains to be clarified.
Finally, a knockout of the siRNA-mediated RhoB mRNA as well as decrease in RhoB (Fig. 7), resulted in the inhibition of ACh release and actin reorganization by LPA (Fig. 8). These results conclusive support our hypothesis that RhoB plays a critical role in neuroexocytosis in PC12 cells.
In summary, our results show that BoNT/A targets RhoB to promote accelerated RhoB degradation by proteasomes, thereby blocking actin dynamics and ACh release. Our data also indicate that a RhoB-related signaling pathway is involved in neuroexocytosis in NGF-treated PC12 cells.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: LPA, lysophosphatidic acid; NGF, nerve growth factor; ACh, acetylcholine; BoNT/A, botulinum toxin type A; SNAP-25, 25-kDa synaptosomal-associated membrane protein; wRhoB, wild-type RhoB; siRNA; small interfering RNA; NSE, neuron-specific enolase; PI, proteasome inhibitor; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; RT-PCR, reverse transcription-polymerase chain reaction; tRhoB, truncated RhoB; FM, fluorescence microscopy; CM, confocal microscopy.
Address correspondence to: Dr. Prabhati Ray, Section of Molecular Biology, Department of Biology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910. E-mail: prabhati.ray{at}na.amedd.army.mil
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