Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid β-peptide-induced apoptosis of PC12 Cells

Authors


Address correspondence and reprint requests to C. M. P. Rodrigues, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Avenue das Forças Armadas, 1600–083 Lisbon, Portugal. E-mail: cmprodrigues@ff.ul.pt

Abstract

Amyloid β-peptide (Aβ)-induced cell death may involve activation of the E2F-1 transcription factor and other cell cycle-related proteins. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates Aβ-induced apoptosis by interfering with crucial events of the mitochondrial pathway. In this study, we examined the role of E2F and p53 activation in the induction of apoptosis by Aβ, and investigated novel molecular targets for TUDCA. The results showed that despite Bcl-2 up-regulation, PC12 neuronal cells underwent significant apoptosis after incubation with the active fragment Aβ (25–35), as assessed by DNA fragmentation, nuclear morphology and caspase-3-like activation. In addition, transcription through the E2F-1 promoter was significantly induced and associated with loss of the retinoblastoma protein. In contrast, levels of E2F-1, p53 and Bax proteins were markedly increased. Overexpression of E2F-1 in PC12 cells was sufficient to induce p53 and Bax proteins, as well as nuclear fragmentation. Notably, TUDCA modulated Aβ-induced apoptosis, E2F-1 induction, p53 stabilization and Bax expression. Further, TUDCA protected PC12 cells against p53- and Bax-dependent apoptosis induced by E2F-1 and p53 overexpression, respectively. In conclusion, the results demonstrate that Aβ-induced apoptosis of PC12 cells proceeds through an E2F-1/p53/Bax pathway, which, in turn, can be specifically inhibited by TUDCA, thus underscoring its potential therapeutic use.

Abbreviations used

amyloid β-peptide

AD

Alzheimer's disease

Apaf-1

apoptosis protease-activating factor 1

CAT

chloramphenicol acetyltransferase

DEVD

N-acetyl-Asp-Glu-Val-Asp

DTT

dithiothreitol

PBS

phosphate-buffered saline

pNA

p-nitroanilide

pRb

retinoblastoma protein

SDS

sodium dodecyl sulfate

TUDCA

tauroursodeoxycholic acid

TUNEL

terminal transferase-mediated dUTP-digoxigenin nick end-labelling

UDCA

ursodeoxycholic acid

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and deficit of cognitive skills. The pathological hallmarks of AD include selective damage to synapses and neurons, neurofibrillary tangles, activated glia and presence of senile plaques (Selkoe 2001). Amyloid β-peptide (Aβ) is the major constituent of senile or amyloid plaques found in the brains of AD patients. Aβ is derived from the processing of the amyloid precursor protein (Haass and Selkoe 1993), and is thought to play a critical role in the onset or progression of AD. Previous studies have shown that Aβ-induced cytotoxicity involves oxidative stress, inflammation and perturbation of calcium homeostasis (Selkoe 2001). In fact, both necrosis and apoptosis are thought to occur in primary neurons and neuronal cell lines after exposure to Aβ, as well as in brains of AD patients (Yankner et al. 1990; Loo et al. 1993; Behl et al. 1994; Su et al. 1994; Mark et al. 1995).

Cell cycle-related molecules are up-regulated in post-mitotic neurons within affected brain regions during AD (McShea et al. 1997; Vincent et al. 1997; Busser et al. 1998). However, it is unclear whether deregulation of cell cycle events contributes to neurodegeneration in AD. E2F-1 is the best characterized member of the E2F family of transcription factors that regulates genes involved in cell cycle, proliferation and apoptosis (Phillips and Vousden 2001). Under certain stress conditions and during the cell cycle, E2F-1 is released from the retinoblastoma protein (pRb) (Mittnacht 1998), thus transactivating its target genes. Interestingly, E2F-1 expression is increased in Aβ-treated cells, suggesting that this transcription factor may also promote neuronal apoptosis via E2F-1 transcriptional activation (Giovanni et al. 1999, 2000; Hou et al. 2000). Activation of E2F-1 induces cells to undergo apoptosis that may occur through stabilization of the tumour suppressor protein p53 via the transcription of p14ARF, transcriptional activation of the p53 homologue p73, and inhibition of the anti-apoptotic signalling of nuclear factor κB (Phillips and Vousden 2001).

Although the role of p53 in suppressing cell cycle progression has been extensively described, less is known about the mechanism by which p53 induces apoptosis. Nevertheless, both E2F-1 and p53 can up-regulate apoptotic proteins such as Bax and the apoptosis protease-activating factor 1 (Apaf-1), resulting in caspase activation and death in several cell types, including neuronal cells (Miyashita et al. 1994; O'Hare et al. 2000; Fortin et al. 2001; Moroni et al. 2001). The involvement of p53 in Aβ-induced apoptosis was initially thought to be negligible (Blasko et al. 2000). However, it has recently been demonstrated that p53 participates in apoptosis of primary human neurons triggered by Aβ peptide, probably through modulation of Bax expression (Zhang et al. 2002). During apoptosis, cytosolic Bax is translocated to the mitochondrial membrane where it induces cytochrome c release. Once in the cytosol, cytochrome c is a co-activator of Apaf-1 in the cleavage of procaspase-9 and execution of apoptosis through the mitochondrial pathway (Green 2000). Exposure of cells to Aβ peptide has been shown to result in mitochondrial perturbation and subsequent caspase activation (Paradis et al. 1996; Selznick et al. 2000; Xu et al. 2001; Luo et al. 2002; Soláet al. 2003a). In addition, we have reported that Aβ induces cytochrome c release via direct mitochondrial membrane permeabilization (Rodrigues et al. 2000a), which appears to be associated with profound changes in membrane lipid and protein structure (Rodrigues et al. 2001).

Ursodeoxycholic acid (UDCA) and its taurine-conjugated derivative TUDCA are endogenous bile acids that increase the apoptotic threshold in several cell types (Rodrigues et al. 1998). We have previously shown that TUDCA stabilizes the mitochondrial membrane and prevents Aβ-induced apoptosis in primary rat neurons (Soláet al. 2003a). TUDCA acts by inhibiting mitochondrial membrane depolarization and channel formation, production of reactive oxygen species, release of cytochrome c and caspase activation (Rodrigues et al. 2000b, 2003a). Interestingly, we have also demonstrated that TUDCA inhibits E2F-1-induced apoptosis, in part, through a caspase-independent mechanism (Soláet al. 2003b). Finally, TUDCA is neuroprotective in a transgenic mouse model of Huntington's disease (Keene et al. 2002), and in rat models of ischemic and haemorrhagic stroke (Rodrigues et al. 2002, 2003b). Here, we further characterized the anti-apoptotic effects of TUDCA in Aβ-induced death of PC12 neuronal cells. Our results suggest that TUDCA specifically inhibits the activation of E2F-1, p53 and Bax triggered by Aβ peptide, thus modulating the apoptotic threshold.

Materials and methods

Cell culture and induction of apoptosis

PC12 cells were grown in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% heat-inactivated horse serum (Sigma Chemical Co.), 5% fetal bovine serum (Invitrogen Corp., Grand Island, NY, USA) and 1% penicillin/streptomycin, and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were plated at either 2 × 105 cells/cm2 for morphological assessment of apoptosis and viability assays, or 4 × 105 cells/cm2 for transfection assays and protein extraction, and differentiated in the presence of nerve growth factor as described previously (Park et al. 1998). PC12 neuronal cells were then incubated in medium supplemented with 100 µm TUDCA (Sigma Chemical Co.), or no addition (control), for 12 h, and exposed to 25 µm Aβ peptide active fragment (25–35) (Bachem AG, Bubendorf, Switzerland) for 1, 8, 24 and 48 h. Cells were fixed for microscopic assessment of apoptosis or processed for cell viability assays. In addition, total and cytosolic proteins were extracted for immunoblotting and caspase activity.

Evaluation of apoptosis and caspase activation

Viability of PC12 neuronal cells was analysed by the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) bromide (Sigma Chemical Co.) and by trypan blue dye exclusion. Hoechst labelling of cells was used to detect apoptotic nuclei. In brief, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min at room temperature, incubated with Hoechst dye 33258 (Sigma Chemical Co.) at 5 µg/mL in PBS for 5 min, and then washed with PBS and mounted using PBS : glycerol (3 : 1, v/v). Fluorescent nuclei were scored blindly by laboratory personnel and categorized according to the condensation and staining characteristics of chromatin. Normal nuclei showed non-condensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous with the nuclear membrane, as well as nuclear fragmentation of condensed chromatin. Three random microscopic fields per sample of approximately 150 nuclei were counted and mean values expressed as the percentage of apoptotic nuclei. In addition, for the terminal transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay, cells were fixed in 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature, and post-fixed in pre-cooled ethanol : acetic acid (2 : 1, v/v) for 5 min at − 20°C. Digoxigenin-nucleotide residues were added to 3′-OH ends of double- or single-stranded DNA by the terminal deoxynucleotidyl transferase. Reactions were performed according to the manufacturer's recommendations (Serologicals Corp., Norcross, GA, USA), and the specimens were then coverslipped with mounting medium before analysis by phase-contrast microscopy.

Finally, caspase activity was determined in cytosolic protein extracts after harvesting and homogenization of cells in isolation buffer containing 10 mm Tris-HCl buffer, pH 7.6, 5 mm MgCl2, 1.5 mm potassium acetate, 2 mm dithiothreitol (DTT) and protease inhibitor cocktail tablets (Complete, Roche Applied Science, Mannheim, Germany). General caspase-3-like activity was determined by enzymatic cleavage of chromophore p-nitroanilide (pNA) from the substrate N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA; Sigma Chemical Co.). The proteolytic reaction was carried out in isolation buffer containing 50 µg cytosolic protein and 50 µm DEVD-pNA. The reaction mixtures were incubated at 37°C for 1 h, and the formation of pNA was measured at 405 nm using a 96-well plate reader.

Transfections and CAT assays

Transfections were performed using reporter constructs E2F-1CAT and 4xE2FCAT, and four expression constructs pCMVE2F-1, pCMVE2F-1▵53, pCMVp53 and pCMVp53(143Ala). E2F-1CAT consisted of the entire human E2F-1 promoter fused to the chloramphenicol acetyltransferase (CAT) gene (Johnson et al. 1993), and 4xE2FCAT was generated by insertion of a synthetic promoter containing four E2F consensus binding sites (Ohtani and Nevins 1994) upstream of the CAT reporter gene. Overexpression plasmids were generated by cloning either wild-type E2F-1 (pCMVE2F-1) and p53 (pCMVp53), or mutant E2F-1 (pCMVE2F-1▵53) and p53 (pCMVp53(143Ala)), all under CMV enhancer/promoter control (Qin et al. 1992). PC12 cells at 40% confluence were transfected with 4 and 8 µg of reporter and expression plasmids, respectively, using Lipofectamine 2000 (Invitrogen Corp.). To assess transfection efficiency, cells were co-transfected with the luciferase construct, PGL3-Control vector (Promega Corp., Madison, WI, USA). Based on this reporter, transfection efficiencies were approximately 70% and did not differ between wild-type and dominant negative plasmids. Twelve hours after E2F-1CAT or 4xE2FCAT transfection, vehicle or 100 µm of TUDCA was added to cells. After an additional 12 h, 25 µm Aβ were included in the cultures. The cells were incubated with Aβ for 24 h, after which all cells were harvested for CAT ELISA (Roche Applied Science) and luciferase assays (Promega Corp.), according to the manufacturers' instructions. Twelve hours prior to transfection with the expression plasmids, PC12 neuronal cells were treated with vehicle or 100 µm TUDCA. At 48 and 60 h post-transfection, all cells were harvested, and total protein extracts were analysed for p53 or Bax expression, respectively. In parallel experiments, cells were also fixed for morphological detection of apoptosis.

Immunoblotting

Steady-state levels of E2F-1, pRb, Mdm-2, p53, Bcl-2, Bax and Bcl-xL proteins were determined by western blot analysis. Briefly, 150 µg total protein extracts were separated on 8 or 12% sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis gels. Following electrophoretic transfer onto nitrocellulose membranes, immunoblots were incubated with 15% H2O2 for 15 min at room temperature. After blocking with 5% milk solution, the blots were incubated overnight at 4°C with primary mouse monoclonal antibodies reactive to E2F-1, Mdm-2, p53, Bax and Bcl-2, or primary rabbit polyclonal antibodies to pRb and Bcl-xS/L (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature. The membranes were processed for protein detection using Super Signal substrate (Pierce, Rockford, IL, USA). β-actin was used as a loading control. Protein concentrations were determined using the Bio-Rad protein assay kit according to the manufacturer's specifications.

Densitometry and statistical analysis

The relative intensities of protein bands were analysed using the ImageMaster 1D Elite version 4.00 densitometric analysis program (Amersham Biosciences, Piscataway, NJ, USA). All data were expressed as mean ± SEM from at least three separate experiments. Statistical analysis was performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA) for the Student's t-test. Values of p < 0.05 were considered significant.

Results

TUDCA inhibits Aβ-induced apoptosis in PC12 cells

Numerous studies have shown that Aβ (1–40) or Aβ (1–42) and its active fragment Aβ (25–35) are highly toxic to primary neurons and a variety of neuronal cell lines. In addition, TUDCA prevents cell death caused by apoptotic stimuli in a multiplicity of cell types (Rodrigues et al. 1998, 2000b; Soláet al. 2003a), suggesting that it could also modulate Aβ toxicity in a neuronal cell line. We used an in vitro model of Aβ-induced death of neuronal PC12 cells, which has provided a potentially valuable insight into Aβ signalling events. Apoptosis was assessed by changes in nuclear and DNA fragmentation, and by caspase-3-like activation (Fig. 1). Increased levels of apoptosis were observed in PC12 cells after incubation with active fragment Aβ (25–35), with a maximum apoptotic response at 48 h (p < 0.01). In contrast, the control reverse peptide Aβ (35–25) was not toxic to PC12 cells (data not shown). TUDCA prevented both nuclear fragmentation and caspase-3-like activation by 60–75% (p < 0.05). A marked protection by TUDCA was also confirmed using cell viability assays (data not shown). Taken together, these findings indicate that PC12 neuronal cells undergo apoptosis when exposed to Aβ, which, in turn, is markedly prevented by TUDCA.

Figure 1.

TUDCA inhibits apoptosis induced by Aβ in PC12 neuronal cells. Cells were incubated with 25 µm Aβ (25–35), or no addition (control), ± 100 µm TUDCA for 1, 8, 24 and 48 h. In co-incubation experiments, TUDCA was added 12 h prior to incubation with Aβ. Cells were fixed and stained for microscopic assessment of apoptosis, and cytosolic proteins were extracted for caspase activity as described in Materials and methods. (a) TUNEL staining in cells incubated with Aβ± TUDCA for 24 h. (b) Fluorescence microscopy of Hoechst staining (top) and percentage of apoptosis in cells exposed to Aβ± TUDCA at the indicated times (bottom). (c) DEVD-specific caspase activity in cytosolic fractions after incubation with Aβ± TUDCA. Cells pre-treated with TUDCA showed less nuclear fragmentation and caspase activation compared with Aβ alone (p < 0.05). The results are expressed as mean ± SEM of at least three different experiments. *p < 0.01 from controls at the same time point.

E2F-1 expression and pRb loss are modulated by TUDCA

The E2F-1 transcription factor controls cell cycle progression as well as induction of apoptosis. It has been suggested that E2F-1 participates in Aβ-induced apoptosis of neuronal cells (Giovanni et al. 2000). In addition, TUDCA inhibited Aβ-associated apoptosis in PC12 cells, indicating that E2F-1 could be an important regulatory factor targeted by TUDCA. PC12 cells were transfected with CAT transcription reporter plasmids under the control of the E2F promoters, and incubated with Aβ for 48 h. CAT activity assays showed that Aβ-induced apoptosis was associated with a mild increase in transcription by E2F-1 at 24 h (p < 0.01) (Fig. 2). Pre-treatment with TUDCA caused a significant decrease in Aβ-driven transcriptional activation of E2F-1 (p < 0.05). In contrast, CAT activity was unchanged with the 4xE2F promoter at 24 h, suggesting that Aβ specifically induces E2F-1 within the E2F family (data not shown). Next, we evaluated E2F-1 protein levels by immunoblot analysis to assess whether transcriptional changes were associated with altered protein expression. PC12 cells exhibited a marked increase in E2F-1 protein levels, which was significant at 1 h (2.5-fold, p < 0.05) through to 48 h (3.5-fold, p < 0.01) of incubation with Aβ peptide (Fig. 3a). Notably, TUDCA reduced Aβ-induced E2F-1 up-regulation by approximately 60% (p < 0.05) (Fig. 3b). Thus, Aβ-induced apoptosis in PC12 neuronal cells appears to involve the E2F-1 transcription factor. Interestingly, it appears that E2F-1 is required for reactivation of the cell cycle, which is a prerequisite for Aβ-induced neuronal death (Copani et al. 1999).

Figure 2.

TUDCA effect on E2F-1-mediated transcription in PC12 cells incubated with Aβ. Cells were co-transfected with a CAT transcription reporter plasmid under the E2F-1-dependent promoter E2F-1CAT, and a luciferase control construct as described in Materials and methods. After 12 h, vehicle or 100 µm TUDCA was added to cells. At 24 h, 25 µm Aβ were included in the cultures and cells harvested for the CAT ELISA and luciferase assays. E2F-1-mediated transcription in cells exposed to Aβ for 1, 8, 24 and 48 h (left), or incubated with Aβ± TUDCA for 24 h (right). CAT activity (absorbance/mg protein) was normalized to control luciferase expression, and the results are expressed as mean ± SEM arbitrary units of at least three different experiments. *p < 0.01 from control; †p < 0.05 from Aβ.

Figure 3.

Modulation of E2F-1 and pRb expression in PC12 cells. Cells were incubated with 25 µm Aβ, or no addition (control), ± 100 µm TUDCA, which was added to the incubation media 12 h prior to incubation with Aβ. Total proteins were extracted for western blot analysis as described in Materials and methods. (a) Protein levels of E2F-1 and pRb in cells incubated with Aβ for 1, 8, 24 and 48 h. (b) Protein levels of E2F-1 and pRb in cells exposed to Aβ± TUDCA for 24 h. The results are normalized to endogenous β-actin production and expressed as mean ± SEM arbitrary units of at least four different experiments. §p < 0.05 and *p < 0.01 from control; †p < 0.05 from Aβ.

The retinoblastoma protein regulates both the transactivation and function of the E2F family of transcription factors. Thus, we determined whether Aβ induced E2F-1 activation by modulating pRb function. Indeed, the results showed that Aβ caused a marked loss of pRb of 30–50% throughout the time course of 48 h (p < 0.01) (Fig. 3a). Further, the inhibition of E2F-1 expression by TUDCA was also dependent on changes in pRb expression. TUDCA prevented Aβ-associated loss of pRb by approximately 65% at 24 h (p < 0.05) (Fig. 3b). Therefore, these data suggest that TUDCA reduces E2F-1 activation induced by Aβ peptide not only by directly modulating E2F-1 levels, but also by inhibiting loss of pRb.

TUDCA inhibits Aβ-induced p53 stabilization via the Mdm-2 protein

Many cellular genes contain E2F sites that contribute to transcriptional regulation. The p53/Mdm-2 pathway is a potential target for the E2F-1 transcription factor, which activates p53 through inhibition of the Mdm-2 protein. Immunoblot analysis of total protein extracts showed that Aβ induced a swift and stable increase in p53 levels up to fivefold at 24 h (p < 0.01) (Fig. 4a). This was accompanied by increased activation of p53, since its inhibitor, Mdm-2, was also markedly diminished during Aβ exposure (about 60% at 8 h, p < 0.01). Incubation with TUDCA alone produced no significant changes in p53 and Mdm-2 protein levels (Fig. 4b). However, co-incubation with TUDCA reduced the observed Aβ increase in p53 by 60%, and almost completely inhibited the decrease in Mdm-2 (p < 0.05).

Figure 4.

Effects of TUDCA on Aβ-induced modulation of Mdm-2 and p53. PC12 neuronal cells were incubated with 25 µm Aβ, or no addition (control), ± 100 µm TUDCA. In co-incubation experiments, TUDCA was added to cells 12 h prior to incubation with Aβ. Total proteins were extracted and subjected to western blot analysis as described in Materials and methods. (a) Protein levels of Mdm-2 and p53 in cells incubated with Aβ for 1, 8, 24 and 48 h. (b) Protein levels of Mdm-2 and p53 in cells exposed to Aβ± TUDCA for 24 h. The results are normalized to endogenous β-actin production and expressed as mean ± SEM arbitrary units of at least four different experiments. §p < 0.05 and *p < 0.01 from control; †p < 0.05 from Aβ.

TUDCA modulates Aβ-induced expression of Bcl-2 family proteins

p53 has been shown to regulate the expression of Bcl-2 family proteins (Miyashita et al. 1994). Our data also suggest that E2F-1-mediated p53 stabilization may modulate Bcl-2 family protein expression. Exposure to Aβ led to a prompt increase in pro-apoptotic Bax levels that was maintained throughout the time course (Fig. 5a). At 24 h of incubation, Bax protein production was enhanced more than twofold (p < 0.01). Anti-apoptotic Bcl-2 was also increased after incubation of PC12 cells with Aβ (p < 0.05). In contrast, the anti-apoptotic Bcl-xL protein remained relatively unchanged. Co-incubation with TUDCA reduced Aβ-associated Bax increase by approximately 80% at 24 h (p < 0.05) (Fig. 5b). Similarly, TUDCA prevented Bcl-2 protein changes. These results suggest that TUDCA prevents Aβ-induced changes in Bcl-2 family protein expression, restoring the equilibrium between pro- and anti-apoptotic members.

Figure 5.

Modulation of Bcl-2 family members in PC12 neuronal cells. Cells were incubated with 25 µm Aβ, or no addition (control), ± 100 µm TUDCA that was added to hepatocytes 12 h prior to incubation with Aβ. Total proteins were extracted for western blot analysis as described in Materials and methods. (a) Representative immunoblots of Bax, Bcl-2 and Bcl-xL proteins in cells exposed to Aβ for 1, 8, 24 and 48 h. (b) Immunoblots of Bax, Bcl-2 and Bcl-xL proteins in cells incubated with Aβ ± TUDCA for 24 h. The blots were normalized to endogenous β-actin protein levels.

The E2F-1/p53/Bax pathway is modulated by TUDCA

To further characterize the mechanism by which TUDCA modulates the p53-regulated apoptosis pathway, we investigated its effects within the E2F-1/p53/Bax pathway. PC12 cells were transfected with plasmids to overexpress either wild-type or mutant E2F-1, or p53. In the absence of Aβ, cells transfected with wild-type E2F-1 and p53 showed about 40 and 65% of apoptosis, respectively, compared with only 10 and 19% of cells transfected with the corresponding mutant plasmids (p < 0.01) (Fig. 6a). Interestingly, TUDCA markedly reduced apoptosis after transfection with wild-type plasmids to almost control levels (p < 0.01).

Figure 6.

TUDCA specifically inhibits the E2F-1/p53/Bax pathway. Twelve hours after incubation with 100 µm TUDCA, PC12 cells were transfected with the constructs pCMVE2F-1 and pCMVE2F-1▵53, or with pCMVp53 and pCMVp53(143Ala) plasmids. At 48 and 60 h post-transfection, cells were fixed and stained for morphological detection of apoptosis. In addition, total proteins were extracted and subjected to western blot analysis of p53 and Bax as described in Materials and methods. (a) Percentage of apoptosis in cells exposed to TUDCA and transfected with the constructs pCMVE2F-1 and pCMVE2F-1Δ53 (left), or with pCMVp53 and pCMVp53(143Ala) plasmids (right). The results are expressed as mean ± SEM of at least four different experiments. (b) p53 expression in cells transfected with the constructs pCMVE2F-1 and pCMVE2F-1Δ53 (left), and Bax expression in cells tranfected with pCMVp53 and pCMVp53(143Ala) plasmids (right). The results were normalized to luciferase expression and expressed as mean ± SEM arbitrary units relative to mutant E2F-1 or p53 of at least three different experiments. §p < 0.05 and *p < 0.01 from respective mutant; ‡p < 0.01 from respective wild-type.

Overexpression of E2F-1 led to about a twofold increase in p53 levels, when compared with cells expressing the E2F-1 mutant plasmid (p < 0.05) (Fig. 6b). Pre-treatment with TUDCA completely prevented the increase in p53 induced by E2F-1 (p < 0.01), suggesting a direct effect at the level of E2F-1. This action was supported by a mild transcriptional repression of E2F-1 by TUDCA (data not shown). However, because overexpressed wild-type E2F-1 is regulated differently from native E2F-1, the protective effect of TUDCA could involve other mechanisms, such as pRb modulation. Cells overexpressing wild-type p53 showed a twofold direct increase in target protein Bax relative to cells expressing mutant p53 (p < 0.05). p53 overexpression was also accompanied by an increase in its degradation, detected by several intense bands with a molecular weight < 53 kDa (data not shown), which may have been because p53 induces its specific inhibitor, Mdm-2. Nevertheless, TUDCA completely prevented the p53-induced Bax expression (p < 0.01). Thus, our results indicate that TUDCA controls p53 function directly, and not only by interfering with upstream factors of the E2F-1/p53/Bax pathway.

Discussion

The precise molecular mechanisms responsible for AD-associated neurodegeneration are not fully understood. However, it has been proposed that Aβ peptide plays a crucial role in the pathogenesis of the disease. Aβ-induced toxicity is a multifactorial process that is thought to involve generation of reactive oxygen species, alteration of intracellular calcium homeostasis, mitochondrial perturbation and caspase activation. We have previously reported that TUDCA prevents Aβ-induced apoptosis of cortical neurons by inhibiting the mitochondrial pathway of cell death (Soláet al. 2003). The present study provides evidence that Aβ-induced apoptosis of PC12 neuronal cells involves activation of the E2F-1/p53/Bax pathway, which, in turn, is significantly altered by TUDCA. Thus, our results suggest that TUDCA can interfere with alternate molecular targets.

Previous studies have reported that a variety of cyclins, cyclin-dependent kinases, and pRb are activated in neurons following exposure to Aβ (Giovanni et al. 1999) and in the brains of AD patients (McShea et al. 1997; Vincent et al. 1997; Busser et al. 1998). Thus, proteins that normally control cell cycle progression in proliferating cells may also modulate neuronal death. Further, neurons lacking E2F-1, a transcription factor regulated by pRb, are significantly protected from death due to Aβ peptide (Giovanni et al. 2000). In the present study, we further investigated the role of E2F-1 in Aβ-induced neurotoxicity. PC12 neuronal cells treated with active fragment Aβ (25–35) showed elevated levels of apoptosis, together with a significant increase in E2F-1 promoter-driven transcription and E2F-1 protein production. The role of E2F-1 in apoptosis was also demonstrated in overexpression experiments, where a significant percentage of cells showed increased nuclear fragmentation. In addition, pRb levels were markedly decreased after Aβ exposure, probably allowing release and activation of E2F-1. This is consistent with previous findings where loss of pRb was associated with increased cell death (Liu and Kitsis 1996; Shan et al. 1996) and overexpression of pRb resulted in enhanced survival (Berry et al. 1996; Fan et al. 1996; Macleod et al. 1996). Finally, mice lacking pRb show massive neuronal loss during development (Jacks et al. 1992).

Activation of E2F-1 may result in modulation of proteins involved in the apoptotic process, such as p53, Bax and Apaf-1, or in the G1/S transition of the cell cycle (Phillips et al. 1997; O'Hare et al. 2000; Moroni et al. 2001). Interestingly, Aβ peptide appears to act as a proliferative signal for differentiated neurons, driving cells into the cell cycle (Copani et al. 1999). It was suggested that neurons must cross the G1/S transition before Aβ induces apoptosis. The mechanism by which Aβ promotes transcription of E2F-1 is still not clear. Nevertheless, TUDCA efficiently prevented E2F-1 up-regulation and pRb loss, thus modulating activity of the transcription factor. This effect may be related to a recently described interaction between unconjugated UDCA and steroid nuclear receptors (Miura et al. 2001), linking cytoprotection to modulation of transcription and gene expression.

The tumour suppressor p53 appears to play an important role in E2F-1-associated apoptosis. In fact, p53 is a tightly regulated molecule, mainly at the level of protein stability. In normal cells, p53 activity is restrained through a negative feedback loop in which the tumour suppressor induces its specific inhibitor, Mdm-2, that, in turn, binds to p53 and targets its proteosomal degradation (Haupt et al. 1997; Kubbutat et al. 1997). However, in cells under stress, p53 is activated in part through inhibition of its degradation by Mdm-2 (Ashcroft et al. 2000). p53 stabilization can also be achieved through an E2F-1-dependent mechanism, in which E2F-1 induces the tumour suppressor protein p14ARF (Bates et al. 1998) that binds to Mdm-2, thus preventing p53 degradation (Kamijo et al. 1998). Interestingly, it has been recently shown that Aβ can promote p53 stability by activating the c-Jun N-terminal protein kinase 1 pathway and increasing the levels of p53, independent of its effect on phosphorylation (Fogarty et al. 2003). Our results indicate that Aβ markedly induced E2F-1 activity, which then resulted in Mdm-2 degradation, p53 stabilization and apoptosis. In contrast, TUDCA significantly suppressed the Aβ peptide effects on p53 and Mdm-2 proteins, thereby inhibiting the Aβ-activated E2F-1/p53 pathway. Further, TUDCA inhibited E2F-1-induced p53 activation and the marked levels of apoptosis with E2F-1 overexpression. Our results suggest that TUDCA inhibition of Aβ-induced apoptosis likely involves pathways for both E2F-1 transcription factor and the tumour suppressor, pRb.

Aβ peptide appears also to modulate protein levels of Bcl-2 family members in PC12 cells, resulting in up-regulation of pro-apoptotic Bax and increased expression of anti-apoptotic Bcl-2. This is not surprising since p53 is known to modulate Bcl-2 family gene expression (Miyashita et al. 1994). p53 is highly expressed in cells after accumulation of Aβ and in the brains of AD patients (LaFerla et al. 1996; Kitamura et al. 1997; Zhang et al. 2002), and p53 inhibitors can prevent neuronal cell death induced by Aβ (Culmsee et al. 2001). Further, Aβ has been shown to either up-regulate pro-apoptotic Bax expression, or require Bax to mediate neurotoxicity (Paradis et al. 1996; Selznick et al. 2000; Culmsee et al. 2001). Although controversial, Bax protein levels have also been reported to increase in AD brain (MacGibbon et al. 1997; Nagy and Esiri 1997; Su et al. 1999). Finally, we have demonstrated that Bax protein increases in mitochondria during Aβ-induced apoptosis of neurons, thus providing a mechanism for cytochrome c release, and subsequent caspase-3 activation and nuclear fragmentation (Soláet al. 2003a). In this study, the results of transgene overexpression suggest that up-regulation of Bax was indeed a result of p53 activation. TUDCA, by decreasing E2F-1 transcriptional activation, prevented the downstream events of Aβ-induced cell death associated with Bax production. Further, TUDCA can specifically modulate the E2F-1/p53/Bax pathway, abrogating E2F-1-induced p53 and p53-associated Bax expression. Of note is the fact that phosphorylated Bad was up-regulated following Aβ incubation (data not shown), thereby possibly sequesting Bad from its mitochondrial targets and increasing the availability of Bcl-2.

In conclusion, this study suggests that TUDCA strongly abrogates Aβ-induced apoptosis of PC12 neuronal cells. The bile acid specifically inhibited the E2F-1/p53 apoptotic pathway, thus modulating the expression of Bcl-2 family elements. The mechanism(s) by which TUDCA regulates the activation of E2F-1, or modulates sections of the E2F-1/p53/Bax pathway, remains to be elucidated. Nevertheless, identification and validation of cellular targets that control life and death decisions may ultimately prove useful when developing new therapeutic interventions for diseases associated with deregulation of the apoptotic process.

Acknowledgements

The authors thank Dr William G. Kaelin, Jr, Harvard University, Boston, MA for the generous gift of pCMV overexpression plasmids. This work was supported by grant POCTI/BCI/44929/2002 from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to CMPR). RMR, SS and REC were recipients of Ph.D. fellowships (SFRH/BD/12641/2003, SFRH/BD/4823/2001 and SFRH/BD/12655/2003, respectively) from FCT.

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