Rho family GTPase inhibition reveals opposing effects of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and Janus kinase/signal transducer and activator of transcription signaling cascades on neuronal survival

Reagents

ToxB and Clostridium sordellii lethal toxin (LTox) were prepared using a method published previously (von Eichel-Streiber et al. 1987). The high-throughput immunoblotting screen was done by BD Pharmingen (Palo Alto, CA, USA) and monoclonal antibodies used for subsequent western blotting of Rac1, c-Raf, ERK1/2, Rab4 and STAT1 were obtained from BD Biosciences (San Diego, CA, USA). Polyclonal antibodies against phospho (p)-PAK1/2/3 (Ser144/141/139), p-MEK1 (Ser298), the dually phosphorylated forms of ERK1 and 2 (p-ERK1 and p-ERK2), and a monoclonal antibody against actin were from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase-linked secondary antibodies and reagents for enhanced chemiluminescence detection were from Amersham Biosciences (Piscataway, NJ, USA). Polyclonal antibody to active caspase 3 was from Promega (Madison, WI, USA). 4,6-Diamidino-2-phenylindole (DAPI), Hoechst dye 33258 and monoclonal antibody against β-tubulin were from Sigma (St Louis, MO, USA). Rabbit polyclonal antibody against Bim was from Stressgen (San Diego, CA, USA). Cy3- and FITC-conjugated secondary antibodies for immunofluorescence were from Jackson Immunoresearch Laboratories (West Grove, PA, USA). PD98059 (PD), U0126 and the small molecule JAK inhibitor I [2-(1,1-dimethylethyl)9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one; JAK Inh] were from Calbiochem (San Diego, CA, USA).

CGN culture

Rat CGNs were isolated from 7-day-old Sprague–Dawley rat pups of both sexes (15–19 g) as described previously (Linseman et al. 2001). Neurons were plated on 35-mm diameter plastic dishes coated with poly-l-lysine at a density of 2.0 × 10⁶ cells/mL in basal modified Eagle's medium containing 10% fetal bovine serum, 25 mm KCl, 2 mm l-glutamine, and penicillin (100 units/mL)/streptomycin (100 µg/mL) (Life Technologies, Grand Island, NY, USA). Cytosine arabinoside (10 µm) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. Using this protocol, the cultures were approximately 95% pure for granule neurons. In general, experiments were performed after 6–7 days in culture.

Cell lysis and immunoblotting

After treatment as described in the results section, cells were kept on ice while the culture medium was aspirated and the cells were washed once with 2 mL ice-cold phosphate-buffered saline (PBS) (pH 7.4). The cells were then incubated for 10 min in lysis buffer (200 µL per 35-mm well) containing 20 mm HEPES (pH 7.4), 1% Triton X-100, 50 mm NaCl, 1 mm EGTA, 5 mmβ-glycerophosphate, 30 mm sodium pyrophosphate, 100 µm sodium orthovanadate, 10 µg/mL leupeptin and 10 µg/mL aprotinin. The cells were then harvested into the buffer by scraping and cell debris was removed by centrifugation at 6000 g for 2 min. A protein assay kit (Pierce Chemical Co., Rockford, IL, USA) was used to determine the protein concentration of the samples. Approximately 80 µg supernatant protein were diluted with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer, boiled for 5 min and electrophoresed through polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences) and processed for immunoblot analysis.

Non-specific binding sites were blocked in PBS (pH 7.4) containing 0.1% Tween 20 (PBS-T) and 1% bovine serum albumin (BSA) for 1 h at room temperature (25°C). The membranes were then incubated for 1 h with primary antibodies diluted in blocking solution. Excess primary antibody was removed by washing the membranes five times with PBS-T over 30 min. Appropriate horseradish peroxidase-conjugated secondary antibodies diluted in PBS-T were then incubated with the membranes for 1 h. Excess secondary antibody was removed with five washes of PBS-T over 30 min. Immunoreactive proteins were detected by enhanced chemiluminescence. In general, western blots shown are representative of the results obtained from three independent experiments. Densitometry was used to quantitate changes in protein expression.

BD Pharmingen Powerblot™

CGNs were incubated with either ToxB (40 ng/mL, final concentration) or vehicle (2 µg/mL BSA in PBS, final concentration) for 24 h and then lysed according to the manufacturer's instructions. Lysates from three different CGN cultures were pooled and sent to BD Clontech for high-throughput immunoblotting against a panel of 1009 purified monoclonal antibodies (BD PowerBlot™). Raw data in the form of image files of the actual blots and densitometric measurements of the immunoreactive proteins were obtained from the manufacturer. The blots shown for Rac1, c-Raf and STAT1 are representative of 2 × 2 comparisons of duplicate control and ToxB lysates.

Quantitation of apoptosis

After treatment, Hoechst dye (8 µg/mL, final concentration) was added directly into the wells for 30 min to stain CGN nuclei. The cells were subsequently washed with 1 mL PBS and then incubated in 4% paraformaldehyde in PBS for 30 min at room temperature. The cells were washed twice more with 1 mL PBS before quantitation. Cells were scored as apoptotic when chromatin condensation or fragmentation was seen. About 300 cells were counted in a minimum of three 60 × fields of a 35-mm dish, and final counts represent data obtained from at least three independent experiments performed in duplicate.

Immunocytochemistry

CGNs were plated at a density of 4.5 × 10⁵ cells per coverslip on glass coverslips coated with polyethyleneimine. After treatment, the cells were fixed in 4% paraformaldehyde, washed once in PBS, and then permeabilized and blocked in 0.2% Triton X-100 and 5% BSA in PBS (pH 7.4). Primary antibodies were diluted in 2% BSA and 0.2% Triton X-100 in PBS. Cells were incubated in the primary antibody for approximately 16 h at 4°C. They were subsequently washed five times in PBS over 30 min and then incubated for 1 h at room temperature with Cy3- or FITC-conjugated secondary antibodies and DAPI diluted in 2% BSA and 0.2% Triton X-100 in PBS. The cells were washed five more times over 30 min with PBS before mounting the coverslips on slides with anti-quench composed of 0.1%p-phenylenediamine in 75% glycerol in PBS. Fluorescent images were captured using a 63 × oil immersion objective on a Zeiss (Thornwood, NY, USA) Axioplan 2 microscope equipped with a Cooke (Romulus, MI, USA) Sensicam deep-cooled charge-coupled device (CCD) camera and a Slidebook software analysis program for digital deconvolution (Intelligent Imaging Innovations Inc., Denver, CO, USA).

Adenovirus preparation and infection

Adenoviral dominant-negative Rho family GTPases were constructed as described previously (Allen et al. 2002). Briefly, Rac1 was cloned from a mouse cDNA library. A dominant-negative (T17N) mutant of Rac1 was generated by PCR-based site-directed mutagenesis. Adenoviral green fluorescent protein (GFP) and GFP/N17Rac1were generated using the AdEasy system (Qbiogene, Inc., Carlsbad, CA, USA). The adenoviruses produced express either GFP alone or in combination with the dominant-negative Rac1 mutant. CGN cultures were infected on day 4 in vitro with adenoviruses at a final titer of 50 infectious particles per cell. Using these conditions, the adenoviruses infected approximately 15–20% of CGNs in the cultures (infection efficiency based on GFP fluorescence detection). At 96 h after infection, cell lysates of infected cells were prepared for immunoblot analysis. Owing to the mechanisms involved in infecting primary neurons, CGNs require at least 48 h for infection to be effective (based on visualization of GFP fluorescence). Therefore, infecting them for 96 h is essentially equivalent to exposing the cells for approximately 48 h to the dominant-negative mutant.

Data analysis

Results represent the mean ± SEM for the number (n) of independent experiments performed. Statistical differences between the means of unpaired sets of data were evaluated by one-way anova, followed by a post hoc Tukey's test. A p-value of < 0.05 was considered statistically significant.

ToxB induces down-regulation of Rac1 and c-Raf in CGNs

ToxB covalently modifies Rho family GTPases by monoglucosylation of a highly conserved threonine residue in the switch 1 region of these G proteins (Just et al. 1995). Addition of a glucose molecule at this site prevents Rho GTPases from interacting with their effectors causing the interruption of downstream signaling pathways (Fig. 1a). ToxB specifically modifies and inhibits Rho, Rac, Cdc42, RhoG and TC10 without affecting other small GTPases such as Ras, Rab or Arf (Just et al. 1995; Just and Gerhard 2004). We have shown previously that incubation of CGNs with ng/mL concentrations of ToxB induces significant apoptosis through a mitochondrial death cascade (Linseman et al. 2001; Le et al. 2005). Consistent with this, CGNs incubated for 24 h with ToxB (40 ng/mL) exhibited a marked increase in fragmented and/or condensed nuclei and significant activation of caspase 3, compared with vehicle-treated CGNs (Fig. 1b). In the present study, we used ToxB to investigate signaling pathways regulated by Rho family GTPases that influence neuronal survival.

To identify proteins that change significantly in expression in response to inhibition of Rho family GTPase function, we initially used a proteomics screening approach. CGNs were incubated with either vehicle or ToxB for 24 h and cell lysates were combined from three separate CGN preparations. Neuronal lysates were then analyzed using the BD PowerBlot™ (BD Pharmingen), a high-throughput immunoblotting screen consisting of more than 1000 monoclonal antibodies to a diverse array of proteins (Malakhov et al. 2003; Li et al. 2004; Kim and Lotan 2005). Independent confirmatory western blots were then performed with new CGN preparations using the same monoclonal antibodies as used in the initial PowerBlot™ screen.

As we recently reported, Rac1 GTPase was markedly decreased in expression in CGNs incubated with ToxB for 24 h (Fig. 2a; upper blots, arrows), whereas Rho and Cdc42 expression did not change under these conditions (see Le et al. 2005). In a similar manner, expression of c-Raf, an upstream serine–threonine kinase in the MAP kinase signaling pathway, was also significantly reduced in CGNs exposed to ToxB (Fig. 2a; lower blots, arrows). Confirmatory western blots verified that the ToxB-induced loss of Rac1 and c-Raf expression observed in the PowerBlot™ screen could be reproduced in a distinct CGN preparation (Fig. 2b). In contrast to these dramatic changes in the expression of Rac1 and c-Raf, the MAP kinases ERK1 and ERK2 showed only minimal decreases in immunoreactivity following ToxB, and the small GTPase Rab4 showed no significant change in expression (Fig. 2b).

Inhibition of Rho family GTPases inactivates a PAK/MEK/ERK1/2 signaling pathway that normally targets pro-apoptotic Bim for degradation

Time course experiments demonstrated that Rac1 immunoreactivity decreased rapidly following exposure to ToxB, with losses of greater than 50 and 75% after 2 and 4 h of Rho family GTPase inhibition respectively (Fig. 3a). Rac and Cdc42 GTPases can each stimulate ERK1/2 signaling via the interaction of their common effector, PAK, with Raf, MEK or ERK kinases (Li et al. 2001; Eblen et al. 2002; Sundberg-Smith et al. 2005). Consistent with Rac/Cdc42 activity regulating a PAK/MEK/ERK1/2 signaling pathway in CGNs, incubation with ToxB rapidly decreased the phosphorylation of PAK, MEK1 and ERK1/2 (Fig. 3b). Interestingly, significant reductions in p-PAK, p-MEK and p-ERK were detected within 1 h of ToxB addition, preceding any detectable loss of Rac1 immunoreactivity (see Fig. 3a). These data suggest that the ToxB-induced glucosylation of Rac/Cdc42 occurs within 1 h and is sufficient to cause a rapid inactivation (dephosphorylation) of the downstream effector PAK. In contrast, a significant loss of Rac1 immunoreactivity was not observed until a slightly later time point, indicating that the degradation of Rac1 is not required for the acute inhibition of downstream PAK signaling.

One recently described pro-survival action of ERK1/2 is phosphorylation of the BH3-only protein, Bim, which targets this pro-apoptotic Bcl-2 family member for degradation via the proteasome (Ley et al. 2003; Luciano et al. 2003). To determine whether ERK1/2 has this pro-survival effect in CGNs, we evaluated the effect of PD, an inhibitor of the upstream kinase MEK, on Bim expression. As expected, incubation of CGNs with PD significantly reduced ERK1/2 activation as shown by decreased phosphorylation of ERK1/2 over an extended period of at least 24 h (Fig. 3c). Moreover, sustained inactivation of ERK1/2 for 24 h with PD resulted in an increase in Bim protein comparable to that observed after 24 h of ToxB exposure (Fig. 3d). These data suggest that ToxB suppresses a Rac/Cdc42-dependent PAK/MEK/ERK1/2 pathway, resulting in decreased proteasomal degradation of Bim. The consequent increase in Bim expression ultimately contributes to CGN death by initiating a mitochondrial apoptotic cascade.

Clostridial toxins with overlapping specificities for inhibiting Rac elicit a JAK-dependent induction of STAT1 and JAK-dependent CGN apoptosis

In contrast to the dramatic reductions in the expression of Rac1 and c-Raf (see Figs 2a and b), inhibition of Rho family GTPases with ToxB induced a marked increase in expression of the transcription factor STAT1 (Fig. 4a). The induction of STAT1 protein following ToxB exposure was largely blocked by co-incubation with actinomycin D (ActD), an inhibitor of gene transcription (Fig. 4b). Moreover, a small molecule pan-JAK inhibitor, JAK Inh (Fig. 4c), also prevented the induction of STAT1 elicited by ToxB (Fig. 4b).

To further establish which Rho family GTPase(s) is involved in regulating STAT1 expression in CGNs, we examined the potential of LTox to induce STAT1. LTox is a monoglucosyltransferase that shows overlapping specificity with ToxB for inhibiting Rac, whereas it inhibits Cdc42 to a much lesser extent and does not inhibit Rho, RhoG or TC10 (Genth et al. 1996; Popoff et al. 1996; Just and Gerhard 2004). Consistent with STAT1 expression being up-regulated in CGNs principally as a result of Rac inhibition, incubation with LTox elicited an increase in STAT1 protein similar to that induced by ToxB (Fig. 4d). STAT1 induction by LTox was similarly blocked by co-incubation with either ActD or JAK Inh. Collectively, these results suggest that inhibition of Rac GTPase leads to stimulation of a JAK/STAT pathway that, in turn, transcriptionally up-regulates STAT1.

STAT1 has recently been shown to play a pro-apoptotic role in the neuronal death induced during ischemic brain injury (Takagi et al. 2002). To determine whether a JAK/STAT pathway exerts a similar pro-apoptotic effect in neurons in which Rho family GTPases (and particularly Rac) are inhibited, we assessed the neuroprotective effects of JAK Inh in ToxB-treated cells. CGNs incubated with ToxB alone demonstrated a marked increase in nuclear condensation and fragmentation relative to vehicle-treated CGNs, and this effect was significantly attenuated by JAK Inh (Fig. 5a). Quantification of apoptosis in CGNs co-incubated with ToxB and JAK Inh revealed significant neuroprotection by the pan-JAK inhibitor at submicromolar concentrations (Fig. 5b). These data indicate that inhibition of Rho family GTPases, and Rac in particular, elicits the activation of a JAK/STAT pathway that plays a significant pro-apoptotic role in CGNs.

The MEK/ERK cascade signals downstream of Rac to repress a pro-apoptotic JAK/STAT pathway in CGNs

The data presented so far indicate that both suppression of the MEK/ERK pathway and induction of a JAK/STAT pathway contribute to CGN apoptosis elicited by inhibition of Rho family GTPases. Next, we investigated whether there is any cross-talk between these two signaling pathways. Several previous studies have shown that ERK activity can negatively regulate JAK/STAT signaling in non-neuronal cells (Jain et al. 1998; Sengupta et al. 1998; Krasilnikov et al. 2003). To determine whether ERK1/2 similarly regulates JAK/STAT signaling in CGNs, we examined the effects of two pharmacological inhibitors of MEK (PD and U0126) on STAT1 expression. Incubation of CGNs for 24 h with PD induced a marked increase in STAT1 protein that was completely blocked by co-incubation with JAK Inh (Fig. 6a). In a similar manner, U0126 significantly reduced ERK1/2 phosphorylation while concomitantly increasing STAT1 expression (Fig. 6b). STAT1 induction elicited by U0126 was completely blocked by JAK Inh; however, the JAK inhibitor had no effect on the loss of p-ERK1/2. These results demonstrate that endogenous MEK/ERK signaling actively represses a JAK-dependent induction of STAT1 in healthy CGNs.

The preceding results show that inhibition of Rho family GTPase function with ToxB results in sequential changes in two signaling pathways that regulate CGN survival. First, inhibition of Rac (and/or Cdc42) function leads to inactivation of a PAK/MEK/ERK1/2 cascade and a consequential decrease in the ERK-dependent degradation of pro-apoptotic Bim. Second, loss of MEK/ERK signaling ultimately results in the de-repression of JAK/STAT activity and induction of pro-apoptotic STAT1. Interestingly, the MEK inhibitors PD and U0126 did not overtly induce CGN apoptosis (data not shown), indicating that the induction of Bim and STAT1 is necessary, but not sufficient, to induce cell death; furthermore, this finding suggests that changes in other pathways downstream of Rho GTPase inhibition are required to initiate apoptosis.

As mentioned previously, we have recently shown that the pro-apoptotic effects of ToxB and LTox in CGNs are largely due to inhibition of Rac GTPase because expression of dominant-negative N17Rac1 is entirely sufficient to mimic the neuronal death elicited by these toxins; furthermore, expression of dominant-negative N17Cdc42 or N19RhoA does not induce CGN apoptosis (Le et al. 2005). To determine whether the above signaling pathways are altered in a similar manner by N17Rac1 and ToxB, we evaluated the effects of adenoviral N17Rac1 on ERK1/2 activity and STAT1 expression. Compared with findings in CGNs infected with adenoviral GFP alone (a negative control), co-expression of GFP and N17Rac1 led to a pronounced inactivation (dephosphorylation) of ERK1/2 and concurrent induction of STAT1 (Fig. 7a). Thus, both the MEK/ERK and JAK/STAT signaling pathways are regulated downstream of Rac activity in CGNs (Fig. 7b).