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The extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase (MAPK) that phosphorylates and regulates various transcription factors in response to growth factors and extra-cellular stresses. To address its biological function during the development of the peripheral nervous system (PNS), we have engineered a novel model of sympathetic neurons in which the erk5 gene can be deleted in vitro. Our data provide for the first time genetic evidence that ERK5 is required to mediate the survival response of neurons to nerve growth factor (NGF). Increased cell death associated with the loss of ERK5 is caused by elevated expression of the BH3-only members of the Bcl-2 family, Bad and Bim. Further investigation indicated that ERK5 suppresses the transcription of the bad and the bim genes via Ca++/cAMP response element binding protein (CREB) and Forkhead box 03a (Foxo3a), respectively. Consistently, we found that the phosphorylation of both p90 ribosomal S6 kinase (RSK) and protein kinase B (PKB) is impaired in neurons lacking ERK5. Together these findings reveal a novel signaling mechanism that promotes neuronal survival during the development of the PNS.
During the development of the brain approximately half of the neurons formed by neurogenesis die by apoptosis before adulthood due to limiting amounts of trophic factors (1). The molecular mechanism underlying this neuronal loss has been greatly facilitated by the phenotypic analysis of nerve growth factor (NGF)-dependent sympathetic neurons lacking specific members of the Bcl-2 family (2). For example, while Bax is essential for NGF withdrawal-induced apoptosis (3), there is functional redundancy between the BH3-only proteins Bad and Bid (4). Unlike bad−/− neurons and bid−/− neurons that do not exhibit any abnormal phenotype, the targeted deletion of bim partially protects sympathetic neurons against trophic factor withdrawal (5). Further studies have shown that increased expression of Bim via the transcription factors c-Jun and Forkhead box 03a (Foxo3a) is critical to trigger neuronal apoptosis (6, 7).
Similarly, de novo protein synthesis is a prerequisite for trophic factor-induced survival. This is exemplified in vivo by the decreased number of sensory and sympathetic neurons in the brain of Ca++/cAMP response element binding protein (creb)−/− mice (8). CREB is a pro-survival transcription factor that acts as both an activator and a repressor of gene expression in the nervous system (9). The transcriptional activity of CREB is increased upon phosphorylation at serine 133 by numerous protein kinases, including p90 ribosomal S6 kinase (RSK), a downstream target of extracellular-regulated protein kinases 1 and 2 (ERK1/2) and ERK5 (10, 11). The specific requirement of ERK5, but not of ERK1/2, to activate CREB in distally stimulated NGF-dependent sensory neurons provided the first evidence that ERK5 was essential for promoting trophic factor-induced neuronal survival (12). The role of ERK5 in mediating the survival of neurons in the central nervous system via the activation of the myocyte enhancer factor 2 (MEF2) transcription factor has since been reported (13, 14).
ERK5 is a non-redundant mitogen-activated protein kinase (MAPK) stimulated in response to growth factors and cellular stresses via the MAPK kinase 5 (MEK5) (15). Since its cloning in 1995, the lack of biological tools, including specific inhibitors, have made it one of the least studied MAPK subfamilies. Consequently, little is known about the downstream targets of ERK5 and therefore the biochemical mechanisms that mediate the effect of ERK5 remain largely unidentified. To advance the current knowledge, we have developed a novel model of primary cultures of sympathetic neurons in which the erk5 gene can be deleted in vitro. Here we provide genetic evidence that ERK5 is required for neuronal survival by suppressing Bad and Bim expression via CREB and Foxo3a, respectively.
The role of ERK5 in NGF-mediated neuronal survival was examined by testing the effect of erk5 gene deletion in sympathetic neurons. Homozygous erk5loxP SCG neurons were infected with an adenovirus encoding Cre recombinase (Cre) or the green fluorescence protein (GFP) (Figure 1a). Immunofluorescence staining confirmed that adenoviruses at 100 multiplicity of infection (MOI) infected neurons with 100% efficiency (Figure 1a). Genomic PCR analysis using specific primers flanking exon 3 revealed that infection with the Cre virus at 100 MOI for 24 h induced efficient recombination of the erk5 gene (Figure 1b). This correlated with the complete loss of the ERK5 protein after 48 h, as observed by immunoblot analysis of the cell lysates using a specific antibody to ERK5 (Figure 1b). The retarded migration of ERK5 following SDS-PAGE analysis of wild type extracts was absent when the cells were incubated with the alkaline phosphatase CIP, suggesting that the upper band detected by immunoblot corresponded to a phosphorylated form of ERK5 (Figure 1c). Similarly, the electrophoretic mobility shift was abolished in SCG neurons cultured in the absence of NGF for 15 and 30 min (Figure 1c). The phosphorylation of ERK5 was restored 30 min after the re-addition of NGF (Figure 1c). Together these results demonstrate that ERK5 is phosphorylated in SCG neurons incubated with NGF.
SCG neurons are dependent on trophic support for their survival. This is demonstrated by NGF withdrawal-induced phosphorylation of the pro-apoptotic c-Jun N-terminal protein kinase (JNK) MAPK (Figure 1d), as well as an increased number of nuclei displaying segmented and condensed chromatin (supplementary Figure 1a and b). In addition, caspase 3 activity was elevated with a maximum at 24 h after NGF deprivation (supplementary Figure 1c). Similarly, the absence of ERK5 for 48 h promoted morphological changes in cell shape (Figure 1e) and in chromatin structure (Figure 1f) typical of apoptotic cells, and significantly increased caspase 3 activity (Figure 1g). However, in contrast to NGF withdrawal, the loss of ERK5 did not increase JNK phophorylation (Figure 1d). The level of apoptotic death associated with ablation of ERK5 in the presence of NGF was comparable to that observed with the removal of NGF for 18 h (Figure 1f and g). Control experiments demonstrated that infection of wild type SCG neurons with the Cre virus was not toxic to the cells (Figure 1h). Together these results indicate that ERK5 is a crucial mediator of the NGF pro-survival signal.
ERK1/2 and protein kinase B (PKB, also known as Akt) have previously been implicated in protecting neurons against stress (17). To establish the relative importance of ERK5, ERK1/2 and PKB in mediating NGF-dependent neuronal survival, we compared the effect of ERK5 deletion with the specific inhibition of ERK1/2 and PKB signaling. Incubation of the cells with either UO126 or wortmannin completely abolished the phosphorylation of ERK1/2 and of PKB at Thr308, but not that of ERK5, demonstrating the specificity of the drugs (Figure 2a). Conversely, ablation of ERK5 by infecting erk5loxP SCG neurons with Cre virus did not affect the phosphorylation of ERK1/2 or PKB at Thr308 (Figure 2a). Under conditions of adequate trophic support, loss of ERK5 or inhibition of ERK1/2 or PKB signaling, increased caspase 3 activity to a similar extent (P>0.05) (Figure 2b). In contrast to our data, several studies have demonstrated that MEK inhibition has minimal effects on NGF-dependent neuronal survival (18-21). Therefore, to confirm our results, we tested the effect of PD0325901, a novel non-competitive inhibitor of MEK1 with greater potency than UO126 (22). Like UO126, we found that PD0325901 specifically inhibited ERK1/2 phosphorylation (Figure 2c) and decreased the survival of SCG neurons incubated with NGF (Figure 2d). Consistent with the caspase 3 assay (Figure 2b) there was no significant difference in cell death exhibited by SCG neurons infected with Cre or incubated with PD0325901 or with wortmannin (Figure 2d). The effect of functional inhibition of ERK5 and ERK1/2, ERK5 and PKB, or ERK5, ERK1/2 and PKB signaling was additive (Figure 2b and d). Together, these results indicate that the ERK5, ERK1/2, and PKB pathways contribute similarly to the survival of NGF-dependent SCG neurons and that neither of them can fully substitute for the loss of the others.
To elucidate the mechanism by which the loss of ERK5 causes neuronal apoptosis we investigated the regulation of Bad, whose functional inhibition by ERK5 prevents apoptosis in endothelial cells (23). Immunoblot analysis using a Bad-specific antibody revealed that the expression of Bad was increased by around 3 fold after ablation of ERK5 (Figure 3a). Immunofluorescence studies confirmed that Bad was expressed at a much lower level in control neurons than in neurons lacking ERK5. Indeed, the exposure time used to generate the images was insufficient to detect Bad in LacZ infected cells (Figure 3b). The staining of Bad displayed by Cre infected erk5loxP neurons partially co-localized with the mitochondrial heat shock protein 70 (mHsp70) (Figure 3b). This is consistent with the pro-apoptotic function of Bad at the mitochondria where it inhibits the activity of anti-apoptotic Bcl-2 family members (24). Similar increases in the expression of the extra long (EL) and long (L) Bim isoforms were detected following the loss of ERK5 (Figure 3c). In contrast, no marked difference was observed in the levels of Bid expression between GFP- and Cre-expressing erk5loxP SCG neurons up until 72 h post-infection (Figure 3d and data not shown). Furthermore, the loss of ERK5 did not cause Bid cleavage (data not shown).
To determine the physiological significance of the increase in bad and bim expression we tested the effect of down-regulating their expression in erk5−/− SCG neurons (Figure 3e and f). Neurons were infected with adenoviruses encoding GFP or Cre 1 h prior to incubating the cells with recombinant shRNA lentivirus targeting the murine bad or bim genes. A recombinant lentivirus encoding unspecific (ctrl) shRNA was used as a control to monitor the effect of lentivirus infection. Immunoblot analysis indicated that Bad and Bim shRNA efficiently and specifically prevented the increase in Bad and Bim expression caused by erk5 gene deletion (Figure 3e). The down-regulation of either of these BH3-only proteins prevented the neuronal death associated with the loss of ERK5 (Figure 3f). Together these results demonstrate that Bad and Bim are required to induce the death of neurons lacking ERK5.
To determine how ERK5 controls Bad and Bim expression, the level of their transcripts in SCG neurons expressing GFP or Cre was measured by quantitative real time (RT) PCR (Figure 4a). The up-regulation of bad mRNA was transient and maximal 18 h after infection with Cre virus, a time which corresponded to a 70% reduction of the erk5 transcript. Expression of Cre for 36 h was necessary to detect increased bim mRNA. Control experiments demonstrated that the transcription of erk5, bad and bim genes was not affected in wild type SCG neurons infected with the Cre virus (data not shown), strengthening our conclusion that ERK5 is required to down-regulate the expression of Bad and Bim under adequate trophic factors conditions.
The transcriptional regulation of Bad and Bim by ERK5 was further examined by luciferase assay using reporter plasmids containing a fragment of the Bad (Bad-Luc) or of the Bim (Bim-Luc) promoter (Figure 4b and c). Increased luciferase activity in SCG neurons following the loss of ERK5 confirmed the negative effect of ERK5 on the bad and bim promoters (Figure 4b and c). Consistent with the quantitative RT PCR time course analysis (Figure 4a), Bad-luc was activated in neurons 18 h after infection, while 36 h was necessary to detect up-regulation of Bim-luc activity.
Increased activity of Bad-luc and Bim-luc was also observed in differentiated PC6.3 over-expressing a dominant negative (DN) form of ERK5 (Figure 4e and f). Immunoblot analysis using a phospho-specific antibody against ERK5 confirmed that ectopic expression of DN-ERK5 blocked the ability of MEK5 to activate ERK5 (Figure 4d). This correlated with increased Bad and Bim expression. The ability of UO126 and wortmannin to activate Bim-luc (Figure 4f), but not Bad-luc (Figure 4e), demonstrated that ERK1/2 and PKB contribute to down-regulating the expression of Bim, but not of Bad, under adequate trophic factor conditions.
A number of response elements have been identified in the promoters of both the bad and the bim genes, including putative binding sites for the transcription factors CREB and Foxo3a, which have both been reported to be downstream targets of the ERK5 signaling pathway (12, 25). Bad(mt)-luc and Bim(dm)-luc reporter constructs carrying deletions of the putative CREB and Foxo3a binding sites respectively, were not responsive to the loss of ERK5 (Figure 4b and c). Together, these results indicated that ERK5 suppresses Bad and Bim expression via its ability to stimulate CREB and to inhibit Foxo3a activity, respectively.
Next we investigated the mechanism by which ERK5 regulated CREB and Foxo3a activity. A previous study showed that activation of ERK5 in sensory neurons promoted the phosphorylation of CREB via RSK (12). Consistent with this result, we found that SCG neurons lacking ERK5 (Figure 5a) or differentiated PC6.3 over-expressing DN-ERK5 (Figure 5b) exhibited impaired phosphorylation of RSK and CREB. Decreased phosphorylation of CREB in SCG neurons was transient with a maximum at 18 h after erk5 gene deletion. This indicates that CREB can be phosphorylated in the absence of ERK5 independently of RSK. This compensatory signaling mechanism could explain the transient up-regulation of the bad transcript in SCG neurons following the loss of ERK5 (Figure 4a).
Chromatin immunoprecipitation (ChIP) analysis using an antibody to CREB demonstrated that CREB interacted with the bad-promoter in differentiated PC6.3 cells incubated with NGF (Figure 5c and d). The DNA purified from the cells was amplified by semi-quantitative (Figure 5c) or by quantitative RT (Figure 5d) PCR. The amount of PCR product was reduced to that of non-specific binding following expression of DN-ERK5, NGF withdrawal, or mutation of the putative CRE sites. Together, these data indicate that ERK5 suppresses Bad expression by stimulating the transcriptional activity of CREB via RSK and by promoting the binding of CREB to the CRE sites in the bad-promoter.
The ability of PKB to prevent increased Bim expression by sequestering Foxo3a in the cytoplasm upon phosphoryation is one mechanism by which PKB promotes neuronal survival (6, 26). In a previous study we have demonstrated that, compared to wild type cells, erk5−/− and mek5−/− fibroblasts treated with sorbitol display reduced PKB activity which is associated with increased Foxo3a activity (25). Here we found that absence of ERK5 in SCG neurons (Figure 6a) or ectopic expression of DN-ERK5 in differentiated PC6.3 (Figure 6b) specifically prevented the phosphorylation of PKB at Ser473, but not at Thr308. Based on evidence that maximal activation of PKB requires dual phosphorylation at Thr308 and Ser473 (27), this result indicates that the level of PKB activity is lower in neurons lacking a functional ERK5 pathway. Consistently, ChIP analysis using an antibody to Foxo3a demonstrated that Foxo3a interacted with the bim-promoter in differentiated PC6.3 cells expressing DN-ERK5 (Figure 6c). Together, these studies suggest that ERK5 down-regulates Bim expression in neurons by a mechanism that implicates PKB-dependent phosphorylation of Foxo3.
This study provides genetic evidence that ERK5 mediates the survival response of developing sympathetic neurons to NGF by suppressing the transcription of both the bim and bad genes (Figure 7). Our hypothesis that ERK5 prevents Bim expression by inhibiting Foxo3a is supported by the demonstration that Foxo transcription factors activate the bim promoter in sympathetic neurons deprived of NGF (6). Our previous study showed that decreased Foxo3a activity in erk5−/− fibroblasts correlated with a reduced PKB activity compared to wild type cells (25). Here, we found that the phophorylation of PKB at Ser473, but not at Thr308, was impaired in neurons 36 h after the loss of ERK5. Together with recent evidence that the phophorylation of PKB at Ser473 is required for PKB to phosphorylate Foxo3a (28), this study indicates that ERK5 down-regulates Bim expression by promoting PKB-dependent inhibition of Foxo3a (Figure 7). It is interesting to note that the inhibition of PKB signaling by wortmannin further increased the level of neuronal death caused by the absence of ERK5. This is consistent with the idea that defective Ser473 phosphorylation affects only a subset of PKB substrates in vivo (28).
Other downstream targets of the ERK5-PKB-Foxo3a signaling pathway include FasL (25). Increased FasL expression enhances apoptosis of ERK5-deficient fibroblasts under conditions of osmotic stress by promoting Bid cleavage (25). In contrast, the death promoting fragment tBid was not detected in erk5−/−neurons. Together with evidence that FasL does not contribute to trophic factor deprivation-induced apoptosis of sympathetic neurons (4), this observation suggests that increased FasL is unlikely to be implicated in the death of SCG neurons associated with the loss of ERK5.
Like Bim, we found that increased Bad expression was critical to trigger neuronal apoptosis following the loss of ERK5. Consistent with our data, a previous study has shown that over-expression of Bad in sympathetic neurons overcomes the survival effect of NGF (29). However, the requirement of Bad to mediate the apoptotic response of neurons caused by the loss of ERK5 appears inconsistent with the functional redundancy of Bad with other BH3-only proteins in NGF withdrawal-induced neuronal death (4). This discrepancy can be explained by the fact that, in addition to ERK5, NGF deprivation inhibits ERK1/2 and PKB activity (17), and stimulates the JNK signaling pathway (30). This leads to additional transcriptional and post-translational modifications of members of the Bcl-2 family which sensitize the cells to apoptotic death. For example, NGF-increased Bcl-2 expression in PC12 cells is blocked following inhibition of ERK1/2 signaling (31). Additionally, upon phosphorylation by JNK, Bim dissociates from the microtubule-associated dynein motor complex and translocates to the mitochondria (32, 33). Thus, mitochondrial translocation of Bim in NGF-deprived neurons exhibiting a low level of Bcl-2 may allow the activation of Bax independently of Bad. This suggests that the transcriptional up-regulation of the bim gene may not be sufficient to trigger neuronal death. Consistent with this, we found that elevated Bim expression in SCG neurons lacking ERK5 and in which the level of Bad is down-regulated, is not toxic to the cells. In conclusion, our results support the idea that Bad and Bim are non-redundant BH3-only proteins unless they are post-translationally modified to increase their pro-apoptotic function.
The mechanism underlying the transcriptional regulation of bad by ERK5 implicates CREB (Figure 7). This result is strengthened by the finding that CREB binds to the putative Cre sites in the bad promoter. CREB activity is regulated by two potential mechanisms. The first is via phosphorylation at Ser133, which increases the transcriptional activity of CREB (34). Evidence that the phosphorylation of CREB and of RSK was impaired in the absence of ERK5 indicates that ERK5 represses bad expression via RSK-dependent activation of CREB. This model is supported by the finding that RSK is a substrate of ERK5 (10) and that ERK5 contributes to mediating CREB phosphorylation following neurotrophin stimulation of sensory neurons (12). Furthermore, the decrease in CREB phosphorylation caused by the loss of ERK5 followed the same transient kinetics as that of the up-regulation of the bad transcript with a maximum after 18 h. The observation that the level of Bad remains elevated up until 48 h after Cre infection suggests that additional mechanisms increase the stability of the protein. The compensatory signaling pathway that partially restores CREB phosphorylation 24 h after the deletion of the erk5 gene, allowing repression of the bad trancription to resume, is unlikely to implicate ERK1/2 considering that the activity of the bad promoter is not affected by UO126. The second mechanism is via the regulation of the binding of CREB to DNA via S-nitrosylation of nuclear proteins that associate with CREB target genes, independently of the phosphorylation of CREB at Ser133 (35). Our findings suggest that ERK5-induced CREB-DNA binding may constitute a mechanism that triggers CREB to act as a repressor of gene expression.
Although ERK5 is required for mediating the survival of sensory (12) and sympathetic (our results) neurons in vitro, mice lacking ERK5 in the brain do not display any obvious developmental defect (36). However, the sympathetic and sensory nervous systems were not specifically examined in the animal model. Therefore, a more thorough phenotypic analysis of the mice lacking ERK5 will be required to firmly conclude on the role of ERK5 during brain development. In contrast, ERK5 was shown to be essential for neural differentiation in Xenopus early embryonic development (37). This discrepancy between Xenopus and mouse models may be explained by the activation of redundant signaling mechanisms in more complex organisms. Although in vitro ERK1/2 and PKB are not able to fully compensate for the loss of ERK5, activation of ERK1/2 and PKB may be sufficient to sustain the survival of neurons lacking ERK5 in mice.
The requirement of PKB to maintain SCG neuronal survival in response to NGF has been reported before (18, 38-40). However one study disputes this conclusion (41). Furthermore, most studies have found that inhibition of MEK has minimal effects on NGF-dependent neuronal survival (18-21). One possible explanation for these controversial findings may lie in the difference in the species from which the neurons were prepared (i.e. rats compared to mice) and in the conditions of the cell cultures. This includes the number of days SCG neurons were kept in vitro prior to experimentation, which can influence the signaling mechanisms in these postmitotic neurons. In addition, while other studies have used PD98059 (18-21), we employed UO126 to block ERK1/2 signaling. To confirm our results, we tested the effect of PD0325901, a novel non-competitive inhibitor of MEK1 with greater potency (22). Like UO126, we found that PD0325901 decreased the survival of SCG neurons incubated with NGF. These conflicting results emphasize the advantage of genetic deletion analyses over the use of selective inhibitors which exhibit variability in their efficiency to specifically block the transduction of signals.
The additive effect of erk5 gene deletion with inhibition of ERK1/2 and PKB signaling in vitro suggests that ERK5, ERK1/2 and PKB are components of independent pathways which contribute to the survival of sympathetic neurons via overlapping mechanisms. For example, while suppression of bad mRNA expression is specifically controlled by ERK5, bim can be transcriptionally regulated by ERK5, ERK1/2 and PKB. In addition, ERK1/2 may be required to maintain Bcl-2 levels (31), and PKB may block the pro-apoptotic function of Bad by phosphorylation (29, 42). The relative importance of these different survival mechanisms to prevent neuronal death is likely to vary depending on the type of stress. For example, ERK1/2, but not PKB, is required to protect SCG neurons against toxic stimuli (43-45). The importance of ERK5 in preventing sympathetic neuronal death due to injury or toxicity remains to be tested. In particular, it will be interesting to determine whether the regulation of the BH3-only protein PUMA, which has been implicated in apoptosis induced by DNA damage in sympathetic neurons independently of JNK (43, 46), is controlled by ERK5. Furthermore, phenotypic analysis of the mice lacking ERK5 in the brain will be required to determine whether ERK5 plays a role in supporting the survival of neurons under certain pathological situations including aging and neurodegenerative diseases.
Sympathetic neurons were obtained from the superior cervical ganglion (SCG) of wild type, erk5+/loxP and erk5loxP/loxP new born mice (post natal day 0-2), as previously described (47). In brief, sympathetic ganglia were trypsinized for 30 min at 37°C. Single-cell suspensions were purified by preplating for 30 min twice on collagen. Non adherent SCG neurons were collected by centrifugation and cultured for 3 days on poly-L-lysine and laminin coated plates in L15 plating medium containing 3% FBS and 50 ng/ml NGF (Alomone Labs), unless indicated otherwise. All mice employed for this study were hosted in a pathogen-free facility at the University Manchester. Use of animals followed Home Office guidelines and received approval by Manchester University's ethical committee.
The PC6-3 subline of the PC12 cell line were cultured on collagen coated plates in differentiating medium (RPMI containing 1% FBS, 2% horse serum, and 100 ng/ml NGF) to obtain a neuronal phenotype, as previously described (6).
SCG neurons were incubated overnight at 55°C with proteinase K. The cell lysates were treated with phenol/TE/hydroxyquinolone and the genomic DNA was isolated by precipitation with isopropanol. Genotype determination was performed by PCR using forward (5′-GCTTCTCCCTGTGATGTGAG-3′) and reverse (5′-TGAGCTACGGGCTTTCG-3′) primers. 1300 bp and 250 bp fragments were amplified from the erk5-flox and disrupted allele, respectively.
The adenoviruses were amplified in HEK-293-T cells and the viral solution was purified on CsCl gradients. The viral infectivity was determined on HEK-293-T cells. After 3 days in culture, SCG neurons were infected with recombinant adenovirus at 100 MOI. Where indicated, recombinant lentivirus at 50 MOI (MissionR shRNA lentiviral transduction particles, Sigma) was added 1 h later. The cells were cultured in plating medium containing 3% FBS and 50 ng/ml NGF for a further 48 h, unless indicated otherwise.
Extracts (15-20 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12%, 10% or 8% polyacrylamide gel) and electrophoretically transferred to an Immobilon-P membrane (Millipore, Inc.). Where indicated, the cell lysate was incubated with CIP for 2 h at 37°C prior to being run on SDS-PAGE. The membranes were incubated with 3% non-fat dry milk at 4°C for 30 min and then probed overnight with antibodies to Bad (BD transduction laboratories), Bim (Calbiochem), Bid (R&D Systems), CREB (Cell Signaling Technology), phospho-CREB at Ser133 (Cell Signaling Technology), ERK1/2 (SantaCruz), phospho-ERK1/2 at Thr202 and Tyr204 (Cell Signaling Technology), ERK5 (Upstate Biotechnology), phospho-ERK5 at Thr218 and Tyr220 (Biosource), JNK (BD Pharmingen), phospho-JNK at Thr183 and Tyr185 (Cell Signaling Technology), PKB (Cell Signaling Technology), phospho-PKB at Thr308 or Ser473 (Cell Signaling Technology), RSK (Cell Signaling Technology), phospho-RSK at Thr359 and Ser363 (Cell Signaling Technology), tubulin (Sigma). Immunecomplexes were detected by enhanced chemiluminescence with anti-mouse or anti-rabbit immunoglobulin G coupled to horseradish peroxidase as the secondary antibody (Amersham-Pharmacia).
The cells were fixed in methanol prior to being incubated with specific antibodies to Cre (Chemicon), to Bad (1:100, BD transduction laboratories) or to the mitochondrial heat shock protein 70 (mHsp70) (1:200, Cell Signaling Technology). Immune complexes were detected with secondary anti-mouse and anti-rabbit antibodies conjugated to Texas red (1:500, Invitrogen) or fluorescein (1:500, Jackson ImmunoResearch), respectively. Nuclei were stained with DAPI (5 μg/ml). Fluorescence images were viewed with an Olympus Widefield microscope.
Wild type and double mutant (dm) Bim-luciferase plasmids were provided by J. Ham (6). Wild type and mutant ERK5 were described before (48). The Bad-luciferase construct was created by subcloning fragment −3599 to −1031 of the murine bad promoter into pGL3-basic vector (Promega) using XhoI and HindIII. A mutant (mt) Bad-luciferase construct carrying deletion of two putative CRE binding sites at −2291 and at −2714 was generated by overlapping PCR.
SCG neurons or PC6.3 cells were transiently transfected using the Metafectene™ reagent (Biontex) with the Bad-, Bim, or CRE-luciferase reporter plasmids together with or without an expression vector encoding DN-ERK5. A pRL-Tk plasmid encoding Renilla luciferase was employed for monitoring transfection efficiency. Aliquots of cell lysates were assayed for firefly and Renilla luciferase activities according to the manufacturer's instructions (Promega).
SCG neurons were incubated with Hoechst 33342 (5 μg/ml) and propidium iodide (5 μg/ml) to distinguish viable, necrotic and apoptotic cells. Between 100 and 200 neurons per conditions were scored for apoptosis using a Leica DMIL microscope (Leica, Wetzlar, Germany). Only neurons that had clearly segmented and condensed chromatin were counted as apoptotic. For caspase assays cells were lysed in 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM DTT, 1 mM EDTA containing 0.1% CHAPS and 10% sucrose. Extracts (5-10 μg) were incubated with 200 μM DEVD-AMC caspase 3 specific fluorogenic substrate for 1 h. Cleavage of the substrate was measured by spectrofluorometer. LDH activity was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay for Promega. Briefly, a 50 μl aliquot was removed from the cell medium and incubated with LDH substrate for 30 min at room temperature in the dark. Stop solution was added to terminate the reaction. The presence of metabolised substrate was measured at absorbance 490nm. The amount of LDH detected was proportional to the amount of lysed cells.
Total RNA was isolated using the Trizol™ reagent and cDNA synthesis was carried out as previously described (49). RT quantitative PCRs were performed using the SYBR Green I Core Kit (Eurogentec). Primers used were: forward primer, 5′-CTGTGTTCTCTGGCACTCCA-3′ and reverse primer, 5′-TCAGCCACACCCAT ATCAAA-3′ for erk5; forward primer, 5′-GCCCCTACCTCCCTACAGAC-3′ and reverse primer 5′-AGGACTTGGGGTTTGTGTTG-3′ for bim; forward primer, 5′- CTCCACATCCCGGAACTCTA-3′ and reverse primer 5′- TTAAAGGGACACAGC GATCC-3′ for bad; and forward primer, 5′-CCAACTTGATGTATGAAGGCTTTG-3′ and reverse primer 5′-AATTGGTCTCAAGTC AGTGTACAGGC-3′ for β-actin to generate amplicons of 100 bp, 84 bp, 107 bp, and 91 bp, respectively. PCR products were detected in the ABI-PRISM 7700 sequence detection systems (Applied Biosystems). Results were analyzed using the 2−ΔΔG methods (50). The level of expression of mRNA was normalized to β-actin mRNA.
PC6.3 cells were transfected with Bad- or Bim-luciferase constructs and subjected to chromatin immunoprecipitation (ChIP) assay (Active Motif) using a CREB antibody (Santa Cruz), a Foxo3a antibody (Upstate Biotechnology), or a control antibody (AKT, cell signaling). Purified immunoprecipitated DNA was subjected to either real time PCR (using the SYBR Green I Core KIT, Eurogentec) or semi-quantitative PCR using the following primer sets: promoter region of bad 5′-TTCCTGAGTGGGCCTCATTCCAGCTG-3′ and 5′-CTGTCCTTACACAGTGCCTTC-3′; promoter region of bim 5′-TGCCACCAAAGATCTCTACC-3′ and 5′-GCATTTCCTCACAGAGTTGG-3′. Input DNA levels served as loading controls for transfection efficiency
We are indebted to B. Carter and A. Tolkovsky for their generosity in providing recombinant adenoviruses. We thank J. Ham for providing us with the PC6.3 cells and the Bim-luciferase constructs and A. Whitmarsh for critically reviewing the manuscript. PD0325901 was a gift from P. Cohen and we thank N. Shpiro for synthesizing the compound. This work was supported by the BBSRC, the Wellcome Trust, and a Lister Institute of Preventive Medicine Research Fellowship to CT.