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Keywords:

  • β-amyloid toxicity;
  • membrane-related oestrogen receptor;
  • mitogen-activated protein kinase;
  • rapid oestrogen signalling;
  • SN56 cell line

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Rapid oestrogen neuroprotection against β-amyloid peptide (Aβ)-induced toxicity, a main feature of Alzheimer's disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17β-oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E-HRP) induced a biphasic stimulation of extracellular-signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4–8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2-related prevention of death in cells exposed to Aβ fragment 1–40 (Aβ1−40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2-associated protection were blocked by an inhibitor of Raf-1 kinase. Raf-1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182 780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf-1/MEK/ERK1/2 pathway, possibly by activation of a membrane-related ER.

Abbreviations used

β-amyloid peptide

AD

Alzheimer's disease

APP

Aβ precursor protein

E2

17β-oestradiol

E-HRP

oestradiol-horseradish peroxidase

ER

oestrogen receptor

MAPK

mitogen-activated protein kinases

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

Studies in different neuronal cell lines have demonstrated that oestrogen (E2) can prevent cell mortality after induction of β-amyloid peptide (Aβ) toxicity (Goodman et al. 1996; Green et al. 1996; Mook-Jung et al. 1997; Marin et al. 2003a). Aβ peptide aggregates within the extraneuronal space forming senile plaques that provoke neurodegeneration and is believed to be one of the primary features in Alzheimer's disease (AD). A consequence of the beneficial effects of oestrogen in this phenomenon is the reduction of Aβ generation and accumulation (Xu et al. 1998; Petanceska et al. 2000; Zheng et al. 2002). These effects seem to be exerted by the hormone through a variety of pleiotropic actions, including its participation in the metabolism of Aβ precursor protein (APP) (Manthey et al. 2001), regulation of APP trafficking from trans-Golgi network (Greenfield et al. 2002) and increase in Aβ uptake by microglia (Li et al. 2000).

Traditionally, it has been sustained that oestrogen elicits neuronal proliferation and survival enhancement by inducing gene transcription upon binding to nuclear oestrogen receptors, α and β (ERα and ERβ). In addition, a variety of recent studies have demonstrated that E2 can exert neuroprotective effects against different injuries through rapid (< 15 min) activation of signalling pathways, such as cAMP/protein kinase A, protein kinase C, and the mitogen-activated protein kinase (MAPK) (Rydel and Greene 1988; Toran-Allerand et al. 1999; Green and Simpkins 2000). In particular, the two isoforms of MAPK, ERK1/2 (extracellular signal-regulated protein kinase), have been shown to be activated by phosphorylation in response to E2, leading to attenuation of neuronal injury during glutamate- and Aβ-induced toxicity (Singer et al. 1999; Fitzpatrick et al. 2002; Mize et al. 2003). However, the potential targets within the plasma membrane that may interact with E2 to elicit the activation of MAPK pathway remain to be clarified. Some studies in several neuronal models have demonstrated the importance of oestrogen receptors in ERK phosphorylation, as observed in hippocampal HT22 cells (Fitzpatrick et al. 2002; Mize et al. 2003). Moreover, a membrane-related ER has been suggested to participate in MAPK cascade stimulation in rat hippocampus (Kuroki et al. 2000) and neocortical explants (Toran-Allerand et al. 2002). In contrast, the ER antagonist ICI 182 780 is unable to attenuate E2-induced ERK activation, as demonstrated in neuroblastoma SK-N-SH cells (Watters et al. 1997) and neocortical explants (Singh et al. 1999). These data indicate that the effects of E2 on stimulating these signalling pathways may depend on the cell type and, most probably, on the model of injury.

In relation to the mechanisms developed by oestrogen to prevent neuronal death during Aβ-induced toxicity, previous data from our group and others have demonstrated the participation of both classical ERs (Kim et al. 2001; Marin et al. 2003a) and a non-classical ER (Marin et al. 2003b). This latter study provided the first evidence of a membrane-related ER involved in this paradigm, although its possible participation in E2-induced activation of signalling pathways was not determined. To date, a single work in hippocampal-derived HT22 cells transfected with ERα and ERβ suggested that both overexpression of these receptors and activation of ERK1/2 phosphorylation by E2 were important events to generate neuroprotection against Aβ-induced toxicity (Fitzpatrick et al. 2002). Even though further studies are required, these preliminary data lead to the hypothesis that alternative ERs may be implicated in neuroprotection by modulating second messenger pathways. Based in our previous data (Marin et al. 2003b), we have used a murine septal cell line, SN56, to ascertain whether the MAPK signalling pathway might participate in rapid effects of E2 to protect against Aβ-induced toxicity via a membrane-related ER.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

The 17β- and 17α-oestradiol, oestradiol bound to horseradish peroxidase (E-HRP), trypan blue and PD98059 were from BioSigma (Madrid, Spain). U0126 and the selective Raf-1 kinase inhibitor I were from Calbiochem (Darmstadt, Germany). ICI 182 780 was a gift from Astra-Zeneca (Madrid, Spain). β-amyloid peptide (fragment 1–40) was obtained from Bachem (St Helens, UK). The monoclonal mouse anti-phospho-p44/p42 MAPK (Thr202/Tyr204) E10 antibody, the polyclonal rabbit anti-p44/42 MAP kinase antibody and the monoclonal rabbit anti-phospho-C-Raf (Ser338) 56A6 antibody were obtained from Cell Signaling Technology (Barcelona, Spain). The monoclonal mouse anti-C-Raf (Raf-1) was obtained from BD Transduction Laboratories (Madrid, Spain). The secondary HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). The Hybond-P transfer membranes, Hyperfilm ECL and the ECL plus Western Blotting Detection System were from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK).

Evaluation of cell death

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

SN56 cells (kindly provided by Dr Bruce Wainer, Wesley Woods Health Center, Atlanta, GA, USA) were cultured at 37°C as previously described (Marin et al. 2001). For treatment with either PD98059 or Raf-1 inhibitor I, cell cultures were grown in serum-free OptiMEM (Gibco Invitrogene, Barcelona, Spain) and pre-incubated with either PD98059 (50 μm), Raf-1 inhibitor I (10 μm) or vehicle [0.1% dimethylsulfoxide (DMSO)] for 30 min. Then, cells were concomitantly exposed for 15 min to the selected inhibitor in the presence of 10 nm of either E2 (diluted in 0.001% ethanol) combined to free HRP (10 nm, diluted in 250 μm Tris-HCl, pH 7.4) or E2 covalently bound to horseradish peroxidase (E-HRP) (diluted in 0.001% ethanol and 250 μm Tris-HCl, pH 7.4). Cultures were washed in OptiMEM and 5 μm1−40 (diluted in 0.05% acetic acid) was added for 24 h. As a control of putative unspecific effects, other cells were exposed to vehicle (0.1% DMSO, 0.001% ethanol and 10 nm HRP diluted in 250 μm Tris-HCl, pH 7.4). In another set of experiments, after hormone exposure for 15 min and washing in OptiMEM, cells were incubated with 5 μm1−40 or vehicle for 6 h, prior to the addition of 50 μm PD98059 for 2 h in the presence of Aβ1−40. Then, the inhibitor was removed and incubation with the amyloid was prolonged for a further 16 h. SN56 viability was determined by trypan blue dye exclusion under the conditions described previously (Marin et al. 2003b). This method has been highly recommended to quantify Aβ-induced cell death (Green et al. 2000).

Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Cell cultures grown in serum-free OptiMEM to 70% confluency were exposed to either 10 nm E2 with free HRP or E-HRP for the indicated times (5–60 min). As a control of unspecific phosphorylation, some cells were exposed to vehicle (0.001% ethanol, 10 nm HRP diluted in 250 μm Tris-HCl pH 7.4). Our preliminary observations indicated that the presence of ethanol, HRP or a combination of both, at the concentrations used here, did not affect basal levels of ERK1/2 phosphorylation (data not shown).

In another set of experiments, cells were exposed to 10 nm of E2 and HRP or E-HRP for 15 min. Then, the steroid was removed and culture incubation in OptiMEM alone was prolonged for different times (0–24 h) prior to protein extraction (see below). For ERK1/2 phosphorylation inhibition assays, cells were pre-exposed to either 50 μm PD98059, 10 μm U0126, 10 μm Raf-1 kinase inhibitor I for 30 min or to 50 μm ICI 182 780 for 2 h. Other cells were concomitantly exposed to the corresponding vehicles. Cell exposure to these molecules was maintained for 15 min more in the presence of 10 nm of either E2 with HRP or E-HRP. Experimental control cultures were exposed to vehicle (10 nm HRP, 0.1% DMSO, 0.001% ethanol). For experiments designed to inhibit the late (8 h) ERK phosphorylation increase, cells previously exposed to either form of the hormone (10 nm, 15 min) or vehicle were washed and incubated in OptiMEM for 6 h. Then, cells were treated with 50 μm PD98059, 10 μm U0126 or the vehicle (0.1% DMSO) for 2 h. After washing out the inhibitors with Tris-buffered saline (TBS) (25 mm Tris-HCl, 137 mm NaCl, 5 mm KCl, pH 7.4), cells were protein extracted (see below).

Immunoblotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

For total protein extraction, cells were scraped with lysis buffer (62.5 mm Tris-HCl, 1% SDS, 10% glycerol, pH 6.8). An aliquot of each extract was preserved for protein quantification by bicinchoninic acid assay (Smith et al. 1985). Five per cent β-mercaptoethanol and 0.001% bromophenol blue were then added and samples were boiled at 95°C for 5 min. Equal amounts (30 μg) of each sample were electrophoresed on 15% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Hybond-P membranes. Membranes were pre-incubated with 5% blotting grade blocker non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) in TBS with 0.1% Tween 20 (TBS-T) at room temperature (20–22°C) for 1 h and washed in TBS-T. To detect ERK1/2, membranes were incubated with a mouse monoclonal specific anti-MAPKs E10 antibody that recognizes these kinases only when residues Thr202 and Tyr204 are phosphorylated, indicating an increase in ERK1/2 activity (Payne et al. 1991). To detect Raf-1 phosphorylation, membranes were incubated with a rabbit monoclonal anti-phospho-Raf-1 antibody that recognizes this kinase only when residue Ser338 is phosphorylated. Phosphorylation of this residue is critical for activation of Raf-1 by Ras kinase (Chong et al. 2001). Membrane incubations with E10 antibody and anti-phospho-Raf-1 antibody [diluted, respectively, 1 : 2000 and 1 : 1000 in TBS-T with 5% bovine serum albumin (BSA)] were performed overnight at 4°C. Antibody-specific labelling was revealed by incubation with a HRP-conjugated goat anti-mouse secondary antibody (1 : 10 000) or a HRP-conjugated goat anti-rabbit (1 : 20 000) and visualized with the ECL chemiluminescence kit (Amersham Biosciences). Membranes were stripped of bound antibodies by incubation in stripping buffer (100 mmβ-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7) at 50°C for 30 min with occasional agitation. Membranes were washed for 3 × 10 min in TBS-T and re-probed with polyclonal rabbit anti-p44/42 MAP kinase antibody or monoclonal mouse anti-Raf-1 antibody (both diluted 1 : 1000 in TBS-T with 5% BSA) overnight at 4°C, followed by incubation with a HRP-conjugated goat anti-rabbit secondary antibody (1 : 20 000) or a HRP-conjugated goat anti-mouse secondary antibody (1 : 10 000). Specific bands were visualized with the ECL chemiluminescence kit, scanned with the GS-800 Calibrated Densitometer and analysed with the image analysis program Quantity One© (Bio-Rad Laboratories). For immunosignal quantification, band intensities were normalized to basal values obtained in vehicle-treated samples. Data were represented as percentage of immunostaining values obtained for either phospho-ERK1/2 or phospho-Raf-1 relative to those obtained for, respectively, total ERK1/2 or Raf-1.

Statistical analyses

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Data were expressed as a mean ± SEM and were analysed by one-way anova followed by Tukey's post-hoc test to compare between groups. Statistical significance is indicated in the figures from p < 0.05. Dose–response curves and time–course analyses were fitted to, respectively, logistic equation and double four-parameter log-normal equations using non-linear regression tools provided in SigmaPlot software package (SPSS Inc., Chicago, IL, USA).

E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Prior studies in several neuronal lines have reported that E2 rapidly activates MAPKs to initiate several intracellular actions, including protection against different insults (Watters et al. 1997; Singer et al. 1999; Fitzpatrick et al. 2002; Mize et al. 2003). In line with this, we have previously reported a membrane-related action of E2 to prevent cell death against Aβ toxicity by still unknown mechanisms (Marin et al. 2003b). Thus, in an attempt to identify the intracellular signalling pathways being involved in this phenomenon, we first investigated in SN56 cells the possible activation by E2 of MAPKs, ERK1 and ERK2. Using a specific antibody that recognizes phosphorylated forms of ERK1/2, we determined by western blot whether exposure for different times (5–60 min) to either E2 or E-HRP may activate ERK1/2 phosphorylation (Fig. 1). A significant increase in phosphorylated forms of these kinases was already observed after 5 min exposure to the steroid (Figs 1a and b, respectively). Maximal phosphorylation was obtained at 15 min whereas no significant levels were detected at 60 min. Basal levels of ERK1/2 phosphorylation in vehicle-treated cells did not vary at the different times of oestrogen exposure (C). Thus, these experimental conditions of maximal phosphorylation of ERK1/2 after 15-min exposure to the hormone were chosen for subsequent assays.

image

Figure 1. 17β-oestradiol (E2) and the membrane-impermeant oestradiol-horseradish peroxidase (E-HRP) rapidly increased extracellular-signal protein kinase phosphorylation in a time-dependent manner. SN56 cells were exposed to 10 nm E2 with HRP (a) or E-HRP (b) for the indicated times (5–60 min). Total cell lysates were collected, and analyzed by western blot using anti-phosphorylated ERK1/2 antibody, or an antibody directed to both phosphorylated and unphosphorylated ERK1/2. Relative levels of phosphorylated ERK2 were determined by densitometric quantification of immunosignal intensities. Values were normalized to the average of basal ones obtained with vehicle-treated cells at the different exposure times (c). Data are represented as percentage of phospho-ERK2 immunoreactivity (IR) values relative to those obtained with total ERK2. *p < 0.05 and #p < 0.0001 versus vehicle-treated cells. Five assays per group.

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In another set of experiments, we characterized the time–course of ERK1/2 phosphorylation during 24 h following 15-min exposure to the steroid (Fig. 2). We observed that, apart from the activation of both forms of MAPKs immediately after hormone treatment, a later phosphorylation peak was observed several hours after treatment with either E2 (Fig. 2a) or E-HRP (Fig. 2b). Analyses of these bimodal patterns using non-linear regression showed that E2 and E2-HRP affected the phosphorylation time–course similarly and following log–normal distributions. The results show maximal phosphorylation times at 23.95 ± 3.66 min and 8.17 ± 0.13 h for E2 (Fig. 2a) and 19 ± 2.66 min and 7.38 ± 0.73 h for E-HRP (Fig. 2b) referred to time 0 at the onset of hormone exposure. R2 values were above 0.97 in all cases. No induction of ERK1/2 phosphorylation was detected in cells exposed to vehicle alone (C). These data indicate that brief oestradiol exposure causes a biphasic induction of ERK phosphorylation. They also suggest that activation of MAPKs pathways may be involved in the response to oestradiol.

image

Figure 2. Short exposures to 17β-oestradiol (E2) or oestradiol-horseradish peroxidase (E-HRP) induce a biphasic pattern of extracellular-signal regulated protein kinase phosphorylation. Cells were exposed to 10 nm E2 with HRP (a) or E-HRP (b) for 15 min, and left to recover in OptiMEM for different times (0 min−24 h). Then, total cell lysates were collected and analyzed by western blot for changes in ERK2 phosphorylation, as described in Fig. 1. Data from E2 and E-HRP-treated cells were fitted to bimodal distributions using log-normal models (depicted as solid lines). (c) Average values of basal levels of phosphorylation detected with vehicle-treated cells at the different times. *p < 0.01 and #p < 0.0001 versus vehicle-treated cells. Six assays per group.

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E2 induces MAPK phosphorylation through MAPK kinase activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

It is known that a variety of extracellular signals produce an activation of MAPK kinases (MEKs) to phosphorylate ERK1/2 without modifying their expression. To study whether enhancement of ERK1/2 phosphorylation by E2 was due to increased MEK activity, we used two compounds, PD98059 and U0126, known to inhibit MEK phosphorylation by Raf. When cells were exposed to different concentrations of PD98059 (0.1–50 μm) for 30 min prior to steroid treatment, we observed that increasing doses of this inhibitor provoked a decrease of ERK phosphorylation, obtaining a total blocking of oestradiol effect at 10–50 μm(Fig. 3a). The estimated IC50 for this inhibition was 7.18 μm, that is close to the IC50 value (2–7 μm) previously described to prevent phosphorylation by Raf of MEK1, whereas higher doses of this compound (IC50 = 50 μm) are required to inhibit MEK2 (Alessi et al. 1995). PD98059 (50 μm) was also observed by western blot to inhibit rapid E2- and E-HRP-induced phosphorylation of ERK1/2 (Fig. 3b). We have confirmed these inhibitory results with U0126 (10 μm), a compound that has been reported to specifically inhibit MEK1 activation by Raf-1 in cells (Davies et al. 2000).

image

Figure 3. MAPK kinase inhibitors, PD98059 and U0126, blocked rapid steroid-induced phosphorylation of extracellular-signal regulated protein kinase (ERK1/2). (a) Concentration–dependence curve for the inhibitory effect of PD98059 on E2-induced ERK phosphorylation in SN56 cells. Cultures were pre-exposed for 30 min to different concentrations (0.1–50 μm) of PD98059 followed by 15 min-treatment with 10 nm oestrogen in the presence of inhibitor. Values of phospho-ERK2 relative to total ERK2 were analyzed by western blot (see Fig. 1). Six assays per group. (b) SN56 cells previously exposed for 30 min to either (50 μm) or U0126 (10 μm), followed by treatment for 15 min with either 10 nm oestrogen (E2) with horseradish peroxidase (HRP) or oestrogen bound to HRP (E-HRP) in the presence of inhibitor. Protein extracts were analysed by western blot. Top: a representative immunoblot assay after incubation of either anti-phospho-ERK1/2 (pERK1/2) antibody or anti-total ERK1/2 antibody. Bottom: densitometric results from immunosignal values of phospho-ERK2 relative to total ERK2. *p ≤ 0.01 and **p≤ 0.0001 versus vehicle-treated cells. Five assays per group.

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Furthermore, the addition of the same doses of either 50 μm PD98059 or 10 µm U0126 6 h after E2 exposure also prevented the second peak of ERK1/2 phosphorylation detected at 8 h after treatment (Fig. 4). The inhibitors did not increase phosphorylation when they were added alone (Figs 3 and 4), indicating that oestradiol was responsible for MEK activation.

image

Figure 4. MAPK kinase inhibitors, PD98059 and U0126, also blocked the late peak of extracellular-signal regulated protein kinase phosphorylation induced by the steroid. SN56 cultures were exposed for 15 min to 10 nm oestrogen (E2) with horseradish peroxidase (HRP) or oestrogen bound to HRP (E-HRP). After oestrogen treatment and washing, culture was prolonged for 6 h more. Then, cells were exposed for 2 h to PD98058 (50 μm) or U0126 (10 μm). Total cell lysates extraction and analyses were performed as described in Fig. 1. Top: a representative immunoblot assay after incubation of either anti-phospho-ERK1/2 (pERK1/2) antibody or anti-total ERK1/2 antibody. Bottom: densitometric results from immunosignal values of phospho-ERK2 relative to total ERK2. *p < 0.0001 versus vehicle-treated cells. Five assays per group.

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Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

We next investigated the possible participation of MAPK signalling cascade in the protective effects of E2 during Aβ toxicity reported previously (Marin et al. 2003b). The 1–40 fragment of Aβ (Aβ1−40), is one of the main molecules found in senile plaques in AD (Iwatsubo et al. 1994). First, we determined the potential effects of Aβ1−40 on ERK phosphorylation in the absence of the hormone. SN56 cultures were exposed for 24 h to either Aβ1−40 (5 μm) alone or in combination with PD98059 (50 μm) in the absence of oestradiol (Fig. 5). These treatments were not observed to affect ERK phosphorylation in SN56 neurons. Then, cell response to 10 nm of either E2 or E-HRP for 15 min, followed by treatment with 5 μm of the 1–40 fragment of Aβ (Aβ1−40) during 24 h, was quantified in the presence of PD98059. Pre-exposure to a 50-μm concentration of PD98059 for 30 min, which blocks the rapid ERK1/2 phosphorylation shown in Fig. 3, also abolished the ability of the steroid to prevent cell death after Aβ1−40 treatment (Fig. 6a). Similar results were obtained when, after pre-treatment, the inhibitor was maintained for 24 h, together with the amyloid peptide. In contrast, no effects on neuroprotection were observed when PD98059 (50 μm) was added 6 h after oestrogen exposure to selectively prevent the late ERK1/2 phosphorylation increase (Fig. 6b). In contrast, PD98059 did not show any significant effect, either when exposed alone or in the presence of the amyloid, indicating that the inhibitor did not interfere with Aβ-related injury (Figs 6a and b). These results suggest that membrane-related effects of E2 to induce neuroprotection depend on the immediate activation of ERK1/2 through MEK.

image

Figure 5. Neither Aβ1−40 nor PD98059 showed any significant increase in extracellular-signal protein kinase phosphorylation. SN56 cultures were exposed to 5 μm1−40 or to 5 μm1−40 in the presence of 50 μm PD98059 for 24 h. Total cell lysates were collected, and analyzed by western blot using anti-phosphorylated ERK1/2 antibody (pERK1/2), or an antibody directed to both phosphorylated and unphosphorylated ERK1/2. Relative levels of phosphorylated ERK2 were determined by densitometric quantification of immunosignal intensities. Values were normalized to basal ones obtained with vehicle (10 nm HRP, 0.05% acetic acid, 0.1% DMSO and 0.001% ethanol)-treated cells (first bar on the left). Data are represented as percentage of phospho-ERK2 immunoreactivity (IR) values relative to those obtained with total ERK2. Six assays per group.

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image

Figure 6. Activation of extracellular-signal regulated protein kinase is required to achieve oestrogen-dependent neuroprotection. SN56 cells were treated with either form of oestradiol (E2 or E-HRP) at 10 nm for 15 min, prior to be exposed to 5 μm of 1–40 fragment of β-amyloid (Aβ1−40). Cell viability was measured by trypan blue exclusion 24 h later (see Materials and methods). Two different treatments were applied for PD98059. In a first set of experiments, cells were pre-incubated with 50 μm of the inhibitor for 30 min before exposure to either the steroid or Aβ1−40 (a). In another set of experiments, the inhibitor was added 6 h after steroid treatment. Two hours later, cells were washed, and culture was elongated up to 24 h in the presence of Aβ1−40 (b). As a control of cell injury, some cultures were incubated with the vehicle, MEK inhibitor or Aβ1−40 alone (first bars on the left). Vehicle-treated cells were used as an experimental control of non-specific cell death. *p < 0.001 versus vehicle-treated cells. Five assays per group.

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Raf-1 kinase phosphorylation at Ser338 is increased by E2

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Some studies have demonstrated that one of the mechanisms accounting for the rapid action of E2 in neurons is through an increase of tyrosine kinase Raf-1 (Watters et al. 2000), which is the upstream enzyme in the MAPK pathway. Raf-1 is known to require phosphorylation of residue Ser338 to be activated (Chong et al. 2001). Therefore, to explore the potential activation of this kinase by oestradiol, we determined by immunoblotting whether 15-min exposure to either E2 or E-HRP may increase Ser338 phosphorylation, using a specific anti-phospho-Raf-1 (Ser338) antibody (Fig. 7). An anti-total Raf-1 antibody was also used as a control of basal levels of Raf-1 expression in SN56 cells. Results show that either form of the steroid produced a significant increase in this phosphorylated form of Raf-1, whereas no phosphorylation was detected in vehicle-treated cells (Fig. 7c).

image

Figure 7. Raf-1 is phosphorylated at Ser338 after short exposure to either 17β-oestradiol (E2) or oestradiol-horseradish peroxidase (E-HRP) in SN56 cells. Cultures were exposed for 15 min to either form of oestradiol (E2 or E-HRP) at 10 nm. Total cell lysates were collected and analyzed by immunoblotting for changes in Raf-1 phosphorylation. Top panel: a representative western blot with anti-phospho-Raf-1 (Ser338) antibody (pRaf-1), showing different levels of band intensities. An antibody to total Raf-1 was used as a control of this protein expression. Lower panel: densitometric analyses of immunoblots obtained with anti-phospho-Raf-1. Values are relative to total Raf-1 immunosignals. *p ≤ 0.001 versus vehicle-treated cells. Six assays per group.

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A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

The increase of Raf-1 activation by oestradiol strongly suggests the involvement of this protein in oestrogen-related signalling at the plasma membrane level. To explore this hypothesis in the present paradigm, cells were first exposed to selective Raf-1 inhibitor I (Lackey et al. 2000) at 10 μm for 30 min prior to oestrogen treatment (Fig. 8a). Interestingly, the presence of the inhibitor was sufficient to completely block ERK1/2 phosphorylation induced by 15-min exposure to either E2 or E-HRP, whereas the inhibitor alone did not have any effect on phosphorylation. These data indicated that E2 enhanced MEK activity to phosphorylate ERK1/2 through activation of Raf-1. Then, it seemed interesting to determine whether Raf-1 may be one of the main factors responsible of E2 neuroprotection at the plasma membrane level. Therefore, we used similar experimental conditions to quantify the effects of Raf-1 inhibitor on E2-dependent cell survival after induction of toxicity by 5 μm1−40 for 24 h (Fig. 8b). Under these conditions, we observed that 30-min exposure to 10 μm of the inhibitor was sufficient to produce a complete blockade of neuroprotection. Moreover, the inhibitor did not show any toxicity in the absence or presence of the amyloid. From these data we can conclude that Raf-1/MEK/MAPK is a main signalling pathway targeted by E2 to elicit neuroprotection against Aβ.

image

Figure 8. The specific inhibitor of Raf-1 kinase blocks oestradiol-induced extracellular-signal regulated protein kinase phosphorylation and neuroprotection. (a) Cultures were exposed to Raf-1 inhibitor I (10 μm) for 30 min prior to be treated with either form of oestradiol (E2 or E-HRP) at 10 nm for 15 min in the presence of the inhibitor. Top panel: a representative western blot with the different anti-ERK1/2 antibodies shows different levels of band intensities obtained for both phospho-ERK1/2 (pERK1/2) and total ERK1/2 with the different treatments. Lower panel: densitometric analyses of immunoblots. Values are relative to total ERK2 immunosignals. (b) After treatment with either Raf-1 inhibitor or the steroid, cells were incubated with 5 μm1−40, and cell viability was quantified by trypan blue exclusion (see Materials and methods). Experimental control cells were incubated in the presence of vehicle, Raf-1 inhibitor or Aβ1−40 alone. Vehicle-treated cells were used as an experimental control of non-specific cell death. *p < 0.001 versus vehicle-treated cells. Five assays per group.

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A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

We have previously observed that the oestrogen receptor antagonist ICI 182 780 (10 µm) partially inhibited (by about 35%) the neuroprotective effect of short exposures (15 min) to either oestradiol or oestradiol conjugated to HRP (10 nm) following Aβ1−40-induced toxicity. These results suggested the existence of an ICI 182 780-sensitive ER that modulates oestrogen-related neuroprotection at the plasma membrane level (Marin et al. 2003b). Here, we wondered whether this receptor might be involved in E2-related activation of Raf-1/ERK/MAPK pathway by using the selective ER antagonist, ICI 182 780. We observed that cell treatment with 10 µm of the anti-oestrogen 2 h before E2 provoked a reduction of the capability of either E2 or E-HRP to increase ERK1/2 phosphorylation, whereas this anti-oestrogen alone did not show any effect on ERK activity (Fig. 9). No phosphorylation induction was obtained when cells were exposed for 15 min at 10 μm of the non-physiological stereoisomer 17α-oestradiol (α-E2). Overall, these results demonstrate that prevention of Aβ-induced cell toxicity takes place by oestrogen interaction with an ICI 182 780-sensitive membrane-related ER and that this association may trigger MAPKs phosphorylation by a mechanism involving activation of both Raf-1 and MAPK kinases.

image

Figure 9. The oestrogen receptor antagonist ICI 182 780 blocks rapid oestrogenic induction of extracellular-signal regulated protein kinase phosphorylation. Cells were first treated for 2 h with an excess (10 μm) of ICI 182 780 and then cultures were co-incubated with both the anti-oestrogen and either form of oestrogen (E2 or E-HRP) for 15 min. Other cells were exposed to the stereoisomer 17α-oestradiol (α-E2) for 15 min. Upper panel: a representative immunoblot obtained with either anti-phospho-ERK1/2 (pERK1/2) antibody or anti-total ERK1/2 antibody. Lower panel: densitometric analyses of immunoblots as percentage of band intensities to total ERK2. *p < 0.05 and **p < 0.0001 versus vehicle-treated cells. Five assays per group.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References

Some clinical studies have indicated that oestrogens may protect against various neurological diseases such as AD (Wise 2003). These observations have been supported by animal and in vitro studies using paradigms that imitate AD pathology, such as Aβ peptide-induced neurotoxicity (Green and Simpkins 2000; Garcia-Segura et al. 2001). Oestradiol can palliate multiple aspects of Aβ injury, resulting in the decrease of Aβ formation, secretion and accumulation (Xu et al. 1998; Li et al. 2000; Manthey et al. 2001; Greenfield et al. 2002) through mechanisms that remain to be elucidated.

Previous work demonstrated that E2-dependent protection against Aβ injury may be partially due to gene transactivation through classical ER (Pike 1999; Kim et al. 2001; Marin et al. 2003a). It was also observed that the steroid increased in a dose-dependent manner both the expression and nucleo-cytoplasmic shuttling of ERα (Marin et al. 2003a), suggesting that oestrogenic modulation of canonical ER behaviour may be an additional feature of the cell response. Aside from classical mechanisms through nuclear receptors, an increasing body of data has shown that oestrogens rapidly initiate a variety of intracellular signalling pathways which are important for cell survival and differentiation (Beyer et al. 2002; Cato et al. 2002). Among them, cAMP-dependent protein kinases, PKC and MAPKs signalling cascades have been shown to be mediated by E2 in neuronal cells (Rydel and Greene 1988; Toran-Allerand et al. 1999; Green and Simpkins 2000). In particular, E2-related induction of MAPK pathway has been described in the hippocampus (Bi et al. 2000; Kuroki et al. 2000), HT22 hippocampal cells (Manthey et al. 2001; Fitzpatrick et al. 2002; Mize et al. 2003), neocortical explants (Singh et al. 1999) and basal forebrain primordia (Dominguez et al. 2004). A membrane impermeant form of E2, E-BSA, was also shown to activate this cascade in SK-N-SH neuroblastoma cells (Watters et al. 1997; Stevis et al. 1999). However, very little is known about the relevance of this intracellular pathway in oestrogen-mediated neuroprotection. In a previous work, we reported that brief (15-min) exposure to physiological doses of either E2 or its impermeant counterpart E-HRP were sufficient to protect a septal cell line (SN56) against Aβ1−40-induced toxicity, suggesting the involvement of an unconventional signalling pathway in this phenomenon (Marin et al. 2003b). Using similar experimental conditions, we have demonstrated here that both forms of the hormone exert a rapid and transient activation of ERK1/2 phosphorylation and that this action takes place through upstream stimulation of MEK, the specific activator of MAPKs (Marshall 1994). Raf-1, one of the members of Raf serine/threonine kinase family known to phosphorylate MEK, was also shown to participate in the E2-induced ERK signalling cascade. This may be particularly interesting as some previous data in neural tissue have identified B-Raf as the main isoform implicated in ERK signalling activation by oestradiol (Toran-Allerand et al. 1999). Oestrogen was also shown here to elicit serine 338 (Ser338) phosphorylation of Raf-1 which seems to play a critical role in this protein kinase activation (Chong et al. 2001), corroborating the involvement of Raf-1 in oestrogen effects in SN56 cells. In a previous work, oestrogen was also shown to cause the rapid and transient tyrosine phosphorylation of Raf-1 in cultured rat pituitary cells (Watters et al. 2000). We have also provided evidence that Raf-1/MEK/ERK1/2 pathway mediates oestrogen-dependent prevention of cell death from Aβ1−40 injury, as selective inhibitors of either MEK (i.e. PD98059) or Raf-1 blocked this effect. Our findings are in agreement with previous data, that have evidenced the importance of MAPK cascade in oestrogen neuroprotection against various types of injury, such as glutamate toxicity (Singer et al. 1999; Mize et al. 2003), Aβ-induced toxicity (Fitzpatrick et al. 2002), and N-methyl-d-aspartate (NMDA)- and kainate-mediated toxicity (Bi et al. 2000). Recently, sustained Raf-1 kinase induction of ERK phosphorylation was observed to participate in hippocampal HT22 cells protection from serum withdrawal-induced cell death (Rössler et al. 2004). Although other factors may be involved, these results point to the existence of common mechanisms triggered by oestradiol to elicit neuroprotection against different types of toxicity.

Apart from the rapid ERK1/2 activation immediately after E2 treatment, we also detected a second increase of phosphorylation after 8-h treatment with either form of the steroid (E2 and E-HRP). However, this late activation did not seem to participate in neuroprotection, as no effects on cell survival were observed when the PD98059 inhibitor was used to specifically block this second increase of ERK1/2 phosphorylation. Though the significance of this second phosphorylation phase cannot be ascertained from the present data, it is likely that it could be as a result of a long-term effect of the steroid. This effect may provoke a possible enhancement of gene transactivation, which in turn may modify other intracellular events.

Many examples have demonstrated that classical ERs can be found associated with the plasma membrane, where they may modulate intracellular signalling (Cato et al. 2002). In a previous work, we demonstrated the presence of a membrane-related ER that participates in rapid neuroprotective actions of oestrogen against Aβ1−40. This receptor was shown to have structural homologies with ERα and was sensitive to the ER antagonist ICI 182 780 (Marin et al. 2003b). This antagonist was observed here to attenuate ERK cascade phosphorylation induced by E2, indicating that an alternative ER is involved in the activation of this signalling pathway. However, high doses of the non-physiological steroisomer 17α-oestradiol did not have any effect either on the activation of ERK (Fig. 9) or on cell survival during Aβ1−40 treatment (Marin et al. 2003b). This indicates that, under the selected experimental conditions, E2 and E-HRP effects are not related to non-specific interactions of these molecules with unspecific components of the plasma membrane. To our knowledge, these results represent the first demonstration of the participation of a non-classical ER in rapid activation of MAPK cascade to elicit neuroprotection against Aβ injury. Some data in HT22 cells transfected with ERα or ERβ have evidenced that receptor expression to activate MAPK is a requirement to protect neuronal cells from glutamate- and Aβ-induced neurotoxicity (Fitzpatrick et al. 2002; Mize et al. 2003). Moreover, a membrane-related ER that shows homologies with ERα and participates in ERK phosphorylation has also been substantiated in neocortical explants (Toran-Allerand et al. 2002) and in breast cancer cells (Song et al. 2002; Zhang et al. 2002). Although not demonstrated in the present work, non-classical ER subpopulation could modulate rapid oestrogenic actions by its direct interaction with some members of MAPK pathway. Examples sustaining this hypothesis have been reported by Singh et al. (1999) in explants of the cerebral cortex, suggesting the existence of a multimeric complex which consists of an ER, Hsp90 and B-Raf and mediates rapid activation of the MAPK pathway. In addition, Wong et al. (2002) have substantiated the involvement of a novel non-genomic activity modulator of ER to enhance ER interaction with Src tyrosine kinase which leads to ERK phosphorylation. Moreover, phosphorylation through MAPK cascade has been shown to enhance both ER activation (Kato et al. 1995) and ER intracellular distribution (Lu et al. 2002).

In summary, the present work demonstrates that oestradiol rapidly elicits neuroprotection against Aβ-induced toxicity in neuronal cells through activation of Raf-1/MEK/ERK1/2 pathway, via a membrane-related ER. Because activation of nuclear ERs is also required to prevent Aβ-induced cell death (Marin et al. 2003a), we could hypothesize that oestrogen effects through ERs associated with the cell membrane may be ultimately coupled with genomic mechanisms to enhance neuroprotection. Further understanding of these pathways may help to elucidate important aspects of the beneficial role of oestrogens in neurodegenerative disorders.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Evaluation of cell death
  6. Modulation of MAPKs (ERK1/2) and Raf-1 phosphorylation
  7. Immunoblotting
  8. Statistical analyses
  9. Results
  10. E2 and E-HRP activate the phosphorylation of mitogen-activated protein kinases, ERK1/2
  11. E2 induces MAPK phosphorylation through MAPK kinase activity
  12. Oestrogen neuroprotection following β-amyloid peptide toxicity is mediated by rapid activation of MAPKs pathway
  13. Raf-1 kinase phosphorylation at Ser338 is increased by E2
  14. A selective Raf-1 kinase inhibitor disrupts E2-induced ERK1/2 phosphorylation and neuroprotection
  15. A membrane-related oestrogen receptor mediates rapid signalling of MAPK pathway
  16. Discussion
  17. Acknowledgements
  18. References