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Keywords:

  • 17β-estradiol;
  • estrogens;
  • hippocampal cells;
  • neuroprotection;
  • protein kinase Cε;
  • protein kinase C inhibitors

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17β-Estradiol (βE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced βE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (α and β) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (α, δ, ε, and ζ) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, βE2 altered only PKCε; it reduced the activity and membrane translocation of PKCε in a manner that correlated with its protection against glutamate toxicity. Furthermore, βE2 reversed the increased activity of membrane PKCε induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCε activity and membrane translocation.

Abbreviations used
βE2

17β-estradiol

BIM

bis-indolylmaleimide

calcein-AM

calcein-acetoxymethylester

DMSO

dimethylsulfoxide

DTE

Dithioerythritol

PI3K

1-phosphatidylinositol 3 kinase

PKA

protein kinase A

PKC

protein kinase C

PMA

4β-phorbol 12-myristate 13-acetate

RFU

relative fluorescence units

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

We and others have demonstrated that estrogens exert potent neuroprotective effects in tissue culture, as well as in animal models (Green and Simpkins 2000; Brinton 2001; Wise 2003). In vitro studies have shown that both 17β-estradiol (βE2), the naturally occurring potent feminizing estrogen, and 17α-estradiol, the biologically inactive isomer of βE2, reduce toxicity caused by serum deprivation, β-amyloid and exposure to glutamate receptor agonists (see review by Green and Simpkins 2000). In addition, βE2 reduces ischemic lesions in animals subjected to middle cerebral artery occlusion (Yang et al. 2000; Shi et al. 2001) and reduces ethanol withdrawal injury in a rat model (Jung et al. 2003, 2004).

Recent evidence indicates that estrogen's neuroprotective action may involve second messenger signaling pathways such as protein kinase C (PKC) (Jung et al. 2003; Hayashi et al. 2005). PKC is an important regulatory enzyme that is variably expressed and activated throughout the CNS, and is credited with a number of functions including cell cycle regulation, neurotransmission and cellular differentiation (Tanaka and Nishizuka 1994; Kauver 1998). Recently, numerous reports have established a role for PKC and specific PKC isozymes in the process of neuroprotection (Maiese and Boccone 1995; Gressens et al. 1999; Noh et al. 2000).

Although accumulating evidence supports a role for PKC in certain forms of neuroprotection, direct evidence linking the neuroprotective effects of estrogens with its alterations in PKC signaling has been lacking. Previous studies have demonstrated that exposure to estrogens can regulate PKC activity and expression in both in vitro and in vivo models (Cordey et al. 2003; Jung et al. 2003; Hayashi et al. 2005). However, the observed effects of estrogen on PKC are quite varied as a result of the variety of cell types, exposure conditions and methodologies used to assess changes in the various PKC isozymes and their activity.

Using the immortalized hippocampal cell line (HT-22) and glutamate insults, the present study examined whether PKC is involved in glutamate toxicity and estrogen protection. We further focused on the involvement of specific PKC isozymes, in particular PKCε, in the neuroprotective effects of βE2. Our previous study reported that estrogen inhibits the protein levels and activity of PKCε associated with ethanol withdrawal insults (Jung et al. 2003; Jung et al. 2005). PKCε has also been reported to be involved in UV-induced cell death (Chen et al. 1999) and apoptotic neuronal insults (Knauf et al. 1999). To this end, we hypothesized that estrogen counteracts the PKC signaling pathway in an isozyme-specific manner as a part of its neuroprotective mechanisms.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents

βE2 was purchased from Steraloids (Wilton, NH, USA). Bis-indolylmaleimide (BIM) and wortmannin were obtained from Calbiochem-Novabiochem Co. (La Jolla, CA, USA).

Cell culture

The immortalized hippocampal cell line HT-22 was used in experiments employing glutamate-induced toxicity. HT-22 cells were obtained from David Schubert (Salk Institute, San Diego, CA, USA). The HT-22 line was originally selected from HT-4 cells based on glutamate sensitivity. HT-4 cells were immortalized from primary hippocampal neurons using a temperature-sensitive SV-40 T antigen (Morimoto and Koshland 1990). HT-22 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT, USA) and gentamicin (50 µg/mL), at 37°C in an atmosphere containing 5% CO2 and 95% air.

Experimental treatments

HT-22 cells were plated into 96-well tissue culture plates at 5000 cells per well in 100 µL cell culture medium. The following day cells were exposed to glutamate at a concentration of 2.5–10 mm. Some 18–24 h later, cell viability was compared with that of vehicle-treated controls. βE2 stock solutions were prepared at a concentration of 10 mm in dimethylsulfoxide (DMSO). BIM and wortmannin were also prepared as stock solutions in DMSO and diluted to the desired concentration in cell culture medium. Vehicle-treated control cultures were exposed in parallel to the same concentration of DMSO as present in experimental cultures. There were no measurable effects of DMSO vehicle treatment on cell viability under these conditions.

Calcein-acetoxymethylester (calcein-AM) viability assay

Cell viability was quantitated using the membrane-permeant calcein-AM dye (Molecular Probes, Eugene, OR, USA). Calcein-AM is a fluorogenic esterase substrate that easily permeates live cells with esterase activity and membranes. Hydrolysis of calcein-AM by intracellular esterases produces calcein, a strongly fluorescent compound that is well retained in the cell cytoplasm, which enables us to measure relative fluorescence units (RFU). Following removal of the medium from the 96-well plates, cells were rinsed once with phosphate-buffered saline (PBS; pH 7.4), and incubated in a solution of 2.5 µm calcein-AM in PBS. After 20 min, fluorescence was determined using a Bio-Tek FL600 microplate reader (Winooski, VT, USA) with an excitation/emission filter set of 485/530 nm. Cell culture wells treated with methanol served as blanks. The results, obtained in RFU, are expressed as the percentage of vehicle-treated control values. The calcein-AM assay proved to be a rapid, accurate and reliable method for the assessment of glutamate-induced toxicity in HT-22 cells. With this protocol there was a linear relationship between the number of viable cells per well and measured calcein-AM fluorescence when viable cell numbers were between 300 and 5000 cellc per well (r2 = 0.9991). Glutamate-induced HT-22 cell death was only evident following 8–10 h of continuous exposure, consistent with previous studies that used alternative methods of measuring viability such as trypan blue dye exclusion (Behl et al. 1995) or the colorimetric methylthiazol tetrazolium test (Tan et al. 1998; Satoh et al. 2000).

Cell fractionation and western blot protocol for PKC assay

Harvested HT-22 cells were homogenized in a buffer containing 20 mm HEPES, pH 7.4, 2 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm Dithioerythritol (DTE) and 10 µg/mL aprotinin. Cells were sonicated to disrupt cell membranes, and the soluble and pellet fractions were separated by centrifugation at 100 000 g as described previously (Watson et al. 1998). After centrifugation, the supernatant was taken for assay of cytosolic PKC and the rest of the whole-cell lysate was used for membrane PKC assay. Equal amounts of cytosolic and membrane cell proteins (20 µg), as determined by the Bradford method, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA) in a Mini Trans-Blot electrophoresis apparatus (Bio-Rad, Hercules, CA, USA) at 100 V for 1 h using Towbin's buffer [25 mm Tris, pH 8.3, 192 mm glycine and 20% (v/v) methanol]. PKC isozyme-specific antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were diluted in buffer containing 20 mm Tris, pH 7.5, and 0.5 m NaCl. Detection of the immune complex was performed using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG and an enhanced chemiluminescence system (Pierce, Rockford, IL, USA). Western blots were quantified using NIH Image 1.47 software for densitometric analysis (NIH Bethesda, MD, USA).

Determination of PKC activity

PKC activity was determined using an ATP in vitro phosphorylation assay (Watson et al. 1994; Watterson et al. 2002). This assay measures PKC activity, expressed as the difference in phosphorylation of PKC-specific substrates between basal and PKC-activated conditions. Briefly, samples to be assayed were diluted to a protein concentration of 1 µg/µL in buffer containing 10 mm HEPES, 5 mm dithiothreitol and 0.1% Triton X-100. Cytosolic and membrane proteins were subsequently assayed for total PKC activity and PKCε-specific activity. Some 10 µL diluted sample was added to 100 µL of a reaction mixture containing 20 mm HEPES, 0.1 mm CaCl2, 10 mm MgCl2, 0.03% Triton X-100, 25 µm MARCKS-PSD peptide (KKRFSFKKSFKL; Calbiochem), 100 µm unlabeled ATP and 0.0228 mCi/mL [γ-32P]ATP in the presence or absence of phosphatidylserine (1 mg/mL) and 4β-phorbol 12-myristate 13-acetate (PMA) (0.2 mg/mL) at 30°C. After 2 min the reaction was terminated by addition of 50 µL 450 mm H3PO4. A 30-µL aliquot of each reaction was spotted on to Whatman P81 filter disks and washed extensively with 150 mm H3PO4. Filters were then washed in ethanol/H2O (1 : 1) and rinsed in acetone. Filters were dried and counted for 32P radioactivity. PKC-specific activity is reported in pmoles ATP per minute per milligram protein. Values are the mean ± SEM for at least three separate experiments.

To measure PKCε-specific activity, the assay was performed in the absence of CaCl2, and a PKCε-specific substrate peptide (ERMRPRKRQGSVRRRV; Calbiochem) replaced the MARCKS-PSD peptide substrate. In these assays, EGTA (1 mm) replaced the omitted CaCl2 (1 mm).

Statistical analyses

Statistical significance was determined by one-way anova followed by a post hoc Tukey's comparison test. p < 0.05 was considered significant for all experiments. For both the western blot assays and PKC activity determinations, each set of data represents three or more individual assays performed separately, with each sample assayed in duplicate and with each sample containing 6–16 replicate wells. Cell viability and PKC activity data are presented as percentages of control values (no treatment). Values are reported as mean ± SEM. Pearson correlation coefficients were calculated to detect the association between the two properties of E2: enhancement of cell survival and inhibition of membrane translocation of PKCε. Pearson correlation coefficient was suitable in this case because both x (cell viability) and y (PKCε protein levels) were dependent variables (Hines et al. 2000; Kuo et al. 2003).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

βE2-induced protection of HT-22 cells

βE2-mediated protection of HT-22 cells was demonstrated by exposing cells to 2.5, 5 or 10 mm glutamate in the presence of βE2 (1, 5 or 10 µm), which preceded the glutamate insult by 4 h. Cell viability was assessed by calcein fluorescence 18–24 h after the insult, and the data are presented as a percentage of values in cells that were not exposed to glutamate (Fig. 1). In the absence of βE2, glutamate decreased cell viability in a concentration-dependent manner. The glutamate-induced HT-22 cell death is consistent with previous studies that used an alternative viability assessment method (Tan et al. 1998; Satoh et al. 2000). βE2 treatment produced a concentration-dependent increase in HT-22 cell survival after the glutamate insult at each concentration of glutamate examined (p < 0.01). Maximal protection from glutamate-induced cell death was observed at 10 µmβE2. Depending on the severity of the glutamate insult, exposure to 10 µmβE2 increased HT-22 cell survival by 1.8- to 4.1-fold.

image

Figure 1. Effect ofβE2 exposure on glutamate-induced toxicity in HT-22 cells. Cells were exposed 2.5, 5 or 10 mm glutamate in the absence or presence of βE2 (1, 5 or 10 µm). βE2 treatment preceded the glutamate insult by 4 h. Twenty-four hours after the insult, cell viability was assessed by calcein fluorescence. Glutamate decreased cell viability in a concentration-dependent manner and βE2 protected against this. Results (mean ± SEM, n ≥ 12) are expressed as a percentage of fluorescence in vehicle controls (not treated with glutamate) *,^,#p < 0.01 versus corresponding control value (no βE2 treatment) (one-way anova followed by a post hoc Tukey's comparison test).

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The exposure period of βE2 (10 µm) before glutamate treatment was subsequently examined (Table 1). Pre-exposure to βE2 was not necessary to elicit the neuroprotective effects. Indeed, full βE2-induced protection was observed when βE2 was administered at the same time, or even 30 min after, the glutamate (10 mm) treatment.

Table 1.  Timing effects of βE2 exposure on glutamate insults in HT-22 cells
Pre-incubation time (min)RFU (% control)βE2-induced protection (+ βE2/–βE2)
–βE2+ βE2
  1. Values are mean ± SEM. Cells were treated with vehicle (– βE2) or βE2 (10 µm) 240 min and 30 min before glutamate (10 mm) insults. Cells were also treated with vehicle (– βE2) or βE2 at the same time as (0 min) and 30 min after (− 30 min) glutamate insults. Cell viability was assessed by calcein fluorescence 18–24 h after the insult. Data represent the RFU expressed as a percentage of the control value (no treatment group). The treatment with βE2 at all four time points protected against glutamate toxicity, suggesting that a long-term pretreatment is not required for estrogen protection.

24018.7 ± 0.664.1 ± 1.33.43-fold
3023.8 ± 0.662.3 ± 1.12.62-fold
019.2 ± 0.766.1 ± 1.53.44-fold
−3018.4 ± 0.567.7 ± 0.83.68-fold

Effects of protein kinase A (PKA) inhibitor or 1-phosphatidylinositol 3 kinase (PI3K) inhibitor on glutamate-induced toxicity in HT-22 cells

The roles of PKA and PI3K in mediating glutamate toxicity and the neuroprotective effects of βE2 were examined. The concentrations of the PKA inhibitor H-89 used (0.1–1.0 µm), were several times higher than the reported Ki (48 nm) for inhibition of PKA activity, but well below the concentrations that inhibit the activity of other protein kinases, including PKC (Ki = 32 µm). The highest concentration of the PI3K inhibitor wortmannin employed (75 nm) was 15 times higher than the reported IC50 (5 nm) of the inhibitor for PI3K (Arcaro and Wymann 1993). Cells were exposed to H-89 or wortmannin in the presence or absence of 10 µmβE2 for 4 h before the addition of the glutamate (10 mm). Inhibition of PKA by H-89 and inhibition of PI3K by wortmannin had no effects on cell viability, nor did it alter the neuroprotection induced by βE2 in this model (data not shown). These findings support the hypothesis that PKA- or PI3K-mediated signaling is not involved in the mechanism of glutamate-induced toxicity in HT-22 cells, or in the neuroprotection induced by βE2.

Effect of PKC inhibition by BIM on glutamate-induced toxicity

The effect of the PKC inhibitor BIM on glutamate-induced toxicity in HT-22 cells was determined by exposing cells to concentrations ranging from 0.2 to 5.0 µm(Fig. 2). The IC50 for inhibition of PKC by BIM is isozyme specific (Toullec et al. 1991; Martiny-Baron et al. 1993), ranging from 0.008 to 0.21 µm. Cells were exposed to BIM 4 h before the addition of glutamate (5 or 10 mm). Cell viability was then determined 24 h after the insult. BIM treatment protected HT-22 cells from glutamate-induced toxicity in a concentration-dependent manner. The level of protection afforded by BIM depended upon the concentrations of both BIM and glutamate; best protection by BIM was evident at the highest dose of BIM and at a lower dose of glutamate. These data suggest that PKC inhibition protects against glutamate-induced toxicity.

image

Figure 2. Effect of BIM on glutamate toxicity in HT-22 cells. Cells were exposed to BIM (0.2–5.0 µm) 4 h before the glutamate (5 or 10 mm) insult. Cell viability was determined 24 h after the insult. The PKC inhibitor BIM protected against glutamate toxicity in a concentration-dependent manner. Results (mean ± SEM, n ≥ 12) are expressed as percentage of fluorescence in vehicle controls (not treated with glutamate). *p < 0.05, **p < 0.001 versus vehicle-treated control group (one-way anova followed by a post hoc Tukey's comparison test).

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Effect of isozyme-specific inhibition of PKC on glutamate-induced toxicity in HT-22 cells

The PKC inhibitor Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3,4-c) carbazole] selectively inhibits PKC isozymes α and β, but does not affect the kinase activity of PKCδ, PKCε and PKCζ (Martiny-Baron et al. 1993). We examined whether the inhibition of PKCα and PKCβ by Go6976 protects against glutamate toxicity in HT-22 cells. The concentration of Go6976 used was 33–100 times higher than the reported IC50 for the inhibition of PKCα (2.3 nm) or PKCβ (6.2 nm) (Martiny-Baron et al. 1993). Unlike the PKC inhibitor BIM, exposure to Go6976 did not alter the toxicity induced by glutamate exposure. These results suggest that inhibition of PKCα or PKCβ does not mediate the neuroprotection against glutamate-induced toxicity (Fig. 3).

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Figure 3. Effect of Go6976 on glutamate toxicity in HT-22 cells and comparison with βE2 and BIM. Cells were exposed to 10 µmβE2, 1 µm BIM or 0.2 µm Go6976. Go6976 is known to inhibit PKCα and PKCβ, but not PKCδ, PKCε and PKCζ. Viability was determined 24 h after the addition of glutamate (5 mm). βE2 and BIM protected against glutamate toxicity whereas Go6976 had no protective effect. Results (mean ± SEM, n ≥ 6) are expressed as a percentage of fluorescence in vehicle-treated controls (not treated with glutamate). *p < 0.01 versus vehicle-treated control group (one-way anova followed by a post hoc Tukey's comparison test).

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Effect of PKC inhibition on βE2-induced protection of HT-22 cells

In order to assess the effect of PKC inhibition on βE2-mediated cell protection, HT-22 cells were exposed to 10 µmβE2, 1 µm BIM, or to a combination of βE2 and BIM. Cell viability was determined 24 h after addition of glutamate (5 mm). All treatments were added to the cells simultaneously, 4 h before the glutamate insult. Experimental results are reported in Table 2. When BIM exposure was combined with that of βE2, the level of neuroprotection (50.6% increase in survival) was greater than that produced by either agent alone (26.0% for βE2, 21.1% for BIM). Furthermore, the combination of βE2 and BIM produced synergistic protection (greater than addition, e.g. 50.6% > 26% + 21.1%). As shown in Fig. 4, statistical analysis using a t-test indicated that the observed effects of βE2 and BIM combination were greater than the algebraic sum of the individual effects of βE2 and BIM (t = 9.7, 12 d.f., p < 0.001 for the observed effects of βE2 + BIM vs. the algebraic sum of βE2 + BIM effects). This supports the possibility that the neuroprotective pathway of βE2 is connected to that of PKC.

Table 2.  Effects of combined βE2 with BIM exposure on glutamate toxicity in HT-22 cells
TreatmentRFU (% control)Increase in survival (%)*
  1. Values are mean ± SEM. HT-22 cells were exposed to 10 µmβE2, 1 µm BIM, or to a combination of βE2 and BIM. Cell viability was determined 24 h following the glutamate (5 mm) insult. All treatments were added to the cells 4 h before the glutamate insult. Data represent the RFU expressed as a percentage of control value (no treatment group). *With respect to vehicle control. A combination of βE2 and BIM produced greater survival than the sum of either protection alone.

Vehicle19.4 ± 1.0
βE245.4 ± 2.326.0
BIM40.5 ± 0.721.1
βE2 + BIM70.0 ± 1.150.6
image

Figure 4. Comparison between additive effects (algebraic sum) and observed synergistic effects of βE2 with BIM on glutamate toxicity in HT-22 cells. HT-22 cells were exposed to 10 µmβE2 or 1 µm BIM, or to a combination of βE2 (10 µm) and BIM (1 µm). Cell viability was determined 24 h after the addition of glutamate (5 mm). All treatments were added to the cells simultaneously, 4 h before the glutamate insult. Data represent the RFU expressed as a percentage of the control value (no treatment group). Combined treatment with βE2 and BIM produced a synergistic protection, which was greater than the algebraic sum of the protection given by either agent alone (t = 9.7, 12 d.f., *p < 0.001 for the observed synergistic effects of βE2 + BIM vs. the algebraic sum of βE2 and BIM effects, n = 5). Additive effects were calculated by the algebraic sum of increased survival induced by either agent alone.

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Identification and intracellular distribution of PKC isozymes in HT-22 cells

Studies were undertaken to identify and characterize the major PKC isozymes of HT-22 cells. HT-22 cells were shown to contain the PKCα, δ, ε and ζ isoforms, but lacked β II or γ. PKCα (82 kDa) was shown to be distributed almost exclusively to the cytosolic fraction under resting conditions. PKCδ (78 kDa) was also found to be distributed primarily to the cytosolic fraction of HT-22 cells. Both PKCε (90 kDa) and PKCæ (72 kDa) isoforms were found to be distributed similarly between the two cell fractions (Fig. 5).

image

Figure 5. Identification and intracellular distribution of PKC isozymes in HT-22 cells. Cytosolic and membrane samples were analyzed by western blotting using PKC isozyme-specific antibodies to PKCα, PKCδ, PKCε and PKCζ. 1, cytosol fraction; 2, membrane fraction. PKCα (82 kDa) was distributed almost exclusively to the cytosolic fraction under resting conditions. PKCδ (78 kDa) was also found to be distributed primarily to the cytosolic fraction of HT-22 cells. Both the PKCε (90 kDa) and PKCζ (72 kDa) isoforms were fairly equally distributed between the two cell fractions. Owing to variations in antibody affinities and avidities, it was not possible accurately to compare the relative intensities of western blot signals between isozymes and make conclusions regarding their relative expression in the cell.

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Effects of βE2 exposure on PKC isozyme distribution in HT-22 cells

Cells were exposed to βE2 at concentrations ranging from 0.01 to 10 µm for 24 h. Cytosolic and membrane proteins were subjected to SDS–PAGE and isozyme-specific PKC western blotting. Chronic βE2 exposure produced little or no alteration in the expression or distribution of PKCα, δ(Fig. 6) or ζ (data not shown) in these cells. In contrast, the expression and distribution of PKCε was significantly altered by βE2 in a βE2 dose-dependent manner (Fig. 7). A significant change in PKCε immunoreactivity was observed in both cellular fractions at or above 1 µmβE2, the minimum concentration needed for neuroprotection in HT-22 cells (Green et al. 1997a, 2001). The altered distribution of PKCε was characterized by a reduction in the membrane-associated protein (p < 0.05), and a corresponding increase in the soluble form of the enzyme (p < 0.05). The reduction in membrane PKCε inversely correlated (Pearson r = − 0.69, p < 0.001), whereas the increase in cytosolic PKCε positively correlated (Pearson r = 0.78, p < 0.001), with protection induced by βE2 (0, 1 and 10 µm) against glutamate (2.5 mm) toxicity (Fig. 8).

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Figure 6. Effect of chronic βE2 exposure on the expression and distribution of PKCα and PKCδ in HT-22 cells. Cells were exposed to βE2 (0.01, 0.1, 1, or 10 µm) for 24 h. Cytosolic and membrane proteins were subjected to SDS–PAGE and isozyme-specific PKC western blotting. βE2 did not alter the profiles of PKCα (a) or PKCδ (b). Results shown are mean ± SEM (n = 5) of three determinations.

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image

Figure 7. Effect of chronic βE2 exposure on the expression of PKCε in HT-22 cells. Cells were exposed to βE2 (0.01, 0.1, 1 or 10 µm) for 24 h. Cytosolic and membrane proteins were subjected to SDS–PAGE and isozyme-specific PKC western blotting. Results shown are the mean ± SEM of three determinations. βE2 concentration dependently decreased the protein levels of membrane PKCε but increased those of cytosolic PKCε. *p < 0.05, **p < 0.01 versus vehicle-treated control group (one-way anova followed by a post hoc Tukey's comparison test). n = 6.

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image

Figure 8. Correlation between βE2-induced cell survival and βE2-induced cellular distribution of PKCε. Cells were exposed 2.5 mm glutamate in the absence or presence of βE2 (1 or 10 µm). βE2 treatment preceded the glutamate insult by 4 h. Twenty-four hours after the insult, cell viability was assessed by calcein fluorescence. Separate cultures were also prepared for the assessment of cytosolic and membrane protein levels of PKCε after exposure to βE2 (1 or 10 µm) for 24 h. Cytosolic and membrane proteins were subjected to SDS–PAGE and PKCε-specific western blotting. Cell protection against glutamate (2.5 mm) toxicity induced by βE2 (0, 1 and 10 µm) correlated inversely with the protein levels of membrane PKCε and positively with cytosolic PKCε (Pearson r = 0.78, p < 0.001 for cytosolic PKCε; Pearson r = − 0.69, p < 0.001 for membrane PKCε). n = 6.

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Each PKC isozyme is thought to become active when the enzyme is bound to the membrane (Newton 1995). These findings provide evidence that chronic estrogen exposure alters the intracellular distribution and expression of PKCε.

Effects of βE2 exposure on PKC activity in HT-22 cells

To examine the effects of βE2 exposure on PKC-specific activity in the HT-22 cell line, cells were exposed to 10 µmβE2 for 2–24 h. Under both assay conditions (total PKC and PKCε specific), we determined that the in vitro addition of βE2 to the reaction assay, at concentrations from 0.1 to 10 µm, had no demonstrable effect on the phosphorylation assay itself (data not shown). Consequently, any changes in PKC-specific activity were the result of estradiol-induced effects that occurred during exposure of the cells. The specific activities obtained for the vehicle-treated controls are shown in Table 3. The percentage of total activity that was determined to be ε specific was similar in the two cellular fractions, ranging from 30% (membrane) to 40% (cytosol) of total.

Table 3.  Comparison of total and PKCε-specific activity in the cytosol and membrane fractions of HT-22 cells
 PKC-specific activity (pmol ATP/min/mg protein)
CytosolMembrane
  1. Values are mean ± SEM for at least three separate experiments. PKC-specific activity is expressed as the difference between activity in the presence and absence of phosphatidylserine and PMA.

Total10 316 ± 12672696 ± 420
PKCε4139 ± 421814 ± 20
(PKCε/Total) × 100 (%)    40  30

The results of exposure to βE2 (10 µm) on PKC-specific activity in HT-22 cells are shown in Fig. 9(a) (PKCε-specific activity) and Fig. 9(b) (total PKC-specific activity). Exposure to βE2 significantly reduced the phosphorylation activity of PKCε in the membrane fraction of HT-22 cells following exposure for at least 1 h (54% of control; p < 0.01). Membrane-associated PKCε activity was reduced to approximately 32% of vehicle-treated control values within 4 h of the initiation of βE2 exposure. This reduction in membrane-associated PKCε activity persisted with continuous βE2 exposure. No significant effect of βE2 exposure on total PKC-specific activity was observed for the time points analyzed (0.5–24 h). There was, however, a trend towards lower total PKC-specific activity in cells exposed to βE2, with the largest effect observed at the 4-h time point. This latter effect of βE2 on total PKC activity may represent the contribution of PKCε-specific activity to total PKC activity because PKCε represents a large portion of the total PKC (Table 3).

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Figure 9. Effect of chronic βE2 exposure on the activity of PKCε and total PKC in HT-22 cells. Cells were exposed to 10 µmβE2 for the times indicated. PKCε-specific (a) and total PKC (b) activities are expressed as the difference between activity in the presence and absence of phosphatidylserine and PMA, and are reported as a percentage of the control value (100%, dashed line). βE2 started to decrease the activity of membrane PKCε after exposure for 1 h Values are the mean ± SEM for at least three separate experiments for total PKC activity. *p < 0.01, **p < 0.001 versus vehicle-treated control group (100%) (one-way anova followed by a post hoc Tukey's comparison test). n = 6.

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Effect of βE2 on the activity of PKCε after glutamate insults in HT-22 cells

Our final experiments determined whether glutamate activates PKCε-specific activity in a manner that is prevented by βE2 exposure (Fig. 10). HT-22 cells were exposed to vehicle or glutamate (5 mm) alone or in combination with βE2 (10 µm). βE2 was added 4 h before exposure to glutamate. After exposure to glutamate for 24 h cells treated with glutamate alone showed a significantly increased activity of membrane PKCε compared with control cells (no glutamate treatment) or the cells treated with glutamate and βE2. Although neither glutamate alone (177 ± 92% of the control value) nor glutamate combined with βE2 significantly altered the activity of cytosol PKCε, there was a trend for βE2 to increase the activity of cytosolic PKCε (226 ± 128% of the control value) with glutamate insults.

image

Figure 10. Effect of βE2 on glutamate-induced activity of PKCε in HT-22 cells. Cells were exposed to 5 mm glutamate for 24 h in the absence or presence of βE2 (10 µm). βE2 treatment preceded the glutamate insult by 4 h. At the end of glutamate exposure, the activity of PKCε was measured using an ATP in vitro phosphorylation assay. PKCε-specific activities (pmoles ATP per minute minute per milligram protein) in the membrane are reported as a percentage of control values (no glutamate and no βE2 treatment). Values are mean ± SEM. *p < 0.01 versus control group (100%) (one-way anova followed by a post hoc Tukey's comparison test). n = 5.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present study demonstrated that both PKC inhibitors and estrogen protect against glutamate-induced neurotoxicity in HT-22 cells and that estrogen inhibits the activity and the membrane translocation of PKC in an isozyme (PKCε)-specific manner. The involvement of PKCε in this neuroprotection is evident from the finding that glutamate activates membrane-bound PKCε whereas estrogen prevents the activation. Two other second messengers, PKA and PI3K, are known to be influenced by estrogen treatment and may be involved in signaling estrogen neuroprotection (Lindford et al. 2000; Yu et al. 2004). However, neither PKA nor PI3K appear to be involved in estrogen protection against glutamate toxicity in HT-22 cells because the specific inhibitors of PKA (H-89) and PI3K (wortmannin) failed to alter cell viability or affect estrogen protection.

Our data provide evidence that inhibition of PKC is neuroprotective. Previous studies, utilizing various models of neuroprotection, also support the concept that PKC inhibition is neuroprotective. The activation of PKC contributes to glutamate-induced neurotoxicity in primary neurons (Pizzi et al. 1996), whereas the inhibition of PKC protects cells from glutamate-induced toxicity (Felipo et al. 1993). The inhibition of PKC before an insult has also been shown to be cytoprotective against anoxia, glucose deprivation and nitric oxide toxicity (Boniece and Wagner 1993; Maiese and Boccone 1995).

Several mechanisms have been proposed for protection afforded by PKC inhibition. PKC-mediated signaling events (i.e. phosphorylation) may activate nucleases and/or proteases required for cell death pathways (Bertolotto et al. 2000). Alternatively, the inhibition of PKC has been shown to attenuate oxidative neuronal injuries induced by buthionine sulfoximine, H2O2 or a pro-oxidant iron (Goodman and Mattson 1994; Higuchi and Matsukawa 1999; Noh et al. 2000). Directly relevant to this hypothesis, oxidants and antioxidants selectively react with the regulatory and catalytic domains of PKC, resulting in activation and inhibition of PKC respectively (Gopalakrishna and Jaken 2000). Finally, PKC-mediated neuroprotection may also be a consequence of PKC's role in cellular apoptosis. Phorbol esters, which initially activate and subsequently down-regulate PKCs, induce apoptosis and cell protection respectively (McConkey et al. 1989; Araki et al. 1990; Deacon et al. 1997; Li et al. 1999). Taken together, these studies suggest that PKC activation under certain conditions may be a common critical step in a variety of neuronal insults.

In the present study, the neuroprotection induced by the combination of a PKC inhibitor with βE2 produced synergistic protection (greater than the additive effect of either agent alone). This suggests that the pathways of PKC and βE2 interact with each other. To test this possibility, we examined the effects of βE2 on individual PKC isozymes (α, δ, ε and ζ) in HT-22 cells. Among these enzymes, only PKCε was altered by βE2, suggesting an isozyme-specific link between the pathways of PKC and βE2. The isozyme specificity is further supported by our finding that Go6976, which selectively inhibits PKCα and PKCβ but not PKCε (Martiny-Baron et al. 1993), failed to alter cell viability. Moreover, our finding that βE2 reversed the glutamate-induced increase in the activity of membrane PKCε suggests a counteraction between PKCε and βE2 in this neuroprotection. The effects of βE2 on PKCε were more prominent in the membrane than in the cytosol: βE2 suppressed both activity and protein levels of PKCε in the membrane, whereas it increased the protein levels of cytosolic PKCε but did not significantly alter the activity in this cell compartment. The reasons for this are not clear at present and require further investigation.

The effect of estradiol on PKC activity could only be demonstrated in the PKCε-specific assay, as no significant effects on total PKC activity were measured. PKCε contributes a significant portion of the total PKC activity, ranging from 30 to 40% of the total (Table 3). The small reduction in total PKC activity by βE2 is therefore probably due to effects of estrogens on PKCε activity.

When stimulated, inactive PKCs in the cytosol translocate to the membrane or cytoskeletal component of cells, interact with anchoring proteins, and finally phosphorylate designated substrates (Csukai and Mochly Rosen 1999; Mochly-Rosen and Kauvar 2000). Our results indicate that βE2 alters the intracellular distribution of PKCε, but not that of PKCα or PKCδ, in a manner that is consistent with a reduced activity of PKCε. Exposure to βE2 increased the protein levels of PKCε in the cytosol and correspondingly decreased levels in the membrane, suggesting that estradiol suppresses the membrane translocation of PKCε.

The mechanisms by which estrogens alter PKC activity are unclear. It is possible that estrogen deactivates a cell surface receptor for PKC. Binding of PKC to cell surface receptors triggers phosphoinositide breakdown and releases calcium from intracellular stores, both of which mediate the downstream events of PKC (Morley et al. 1992). Alternatively, estrogens may alter PKC activity through antioxidant properties. The HT-22 cell line lacks ionotropic glutamate receptors and so glutamate kills the HT-22 cells by a mechanism not involving excitotoxic glutamate receptors (Zaulyanov et al. 1999). Instead, HT-22 cells contain the glutamate/cystine antiporter, which is required for the delivery of cystine into neuronal cells. Inhibition of cystine uptake by excess extracellular glutamate ultimately leads to a reduction in endogenous antioxidant glutathione and cell death (Murphy et al. 1989; Tan et al. 1998). In agreement with this, glutamate at millimolar concentrations induced oxidative stress in the same cell line (Davis and Maher 1994). At concentrations that suppress the membrane translocation and activity of PKC in HT-22 cells, βE2 demonstrates potent antioxidant activity (Behl et al. 1995; Goodman et al. 1996; Green et al. 1997b). Oxidants selectively react with the regulatory domain to activate PKC, whereas antioxidants appear to interact with the catalytic domain to inhibit cellular PKC activity (Gopalakrishna and Jaken 2000). These findings support the hypothesis that estrogen inactivates PKC, prevents membrane translocation, and thereby protects cells from oxidative insults (Jung et al. 2004, 2005).

Growing evidence suggests that the novel PKC isozymes such as PKCε and PKCδ are generally pro-apoptotic in nature (Cross et al. 2000) and that their inhibition is protective (Carpenter et al. 2002; Petrovics et al. 2002). The activation of PKCε was shown to be required for UV-induced apoptosis (Chen et al. 1999). In contrast, PKCε-negative mutants block apoptosis triggered by a variety of neuronal insults (Knauf et al. 1999). βE2 exerts neuronal protection by increasing the expression of the anti-apoptotic protein Bcl-xL (Pike 1999), by decreasing levels of nip-2 mRNA, which encodes a pro-apoptotic protein (Meda et al. 2000), and by blocking the DNA degradation that accompanies glutamate insults (Behl et al. 1997). Relevant data come from our previous study, in which estrogen was shown to suppress PKCε in ethanol withdrawn rats and attenuate apoptotic cell death in the same rat group (Jung et al. 2003).

On the other hand, in certain models of neuroprotection PKCε has exhibited somewhat controversial effects, such as protection against mild ischemic brain insults (Raval et al. 2003). Cordey et al. (2003) reported that estrogen activates PKC in the cortical neurons to exert protection against β-amyloid toxicity. Although the reasons for these discrepancies are not clear, there are a few explanations. First, the PKC assay used by Cordey et al. (2003) neither discriminated between PKC isozymes nor specifically measured PKCε activity, whereas an isozyme-specific assay was used in our study. Second, they applied low concentrations of estrogen (nanomolar range) to cerebrocortical neurons whereas pharmacological concentrations (nanomolar to micromolar) were applied to hippocampal neurons in our study. In fact, when we examined the effects of βE2 on PKC in the hippocampus and cortex of rats withdrawn from ethanol, we saw a similar phenomenon; E2 increased the activity of PKCε in the cortex but decreased it in the hippocampus (M. E. Jung, unpublished observation). Given this, one might speculate that PKC, in particular PKCε, is a molecular sensor that modulates signal homeostasis depending upon concentration, brain region or the nature of insults.

In summary, we assessed PKCs to determine their roles in estrogen protection. Inhibition of two other protein kinases (PKA and PI3k) failed to alter cell viability regardless of estrogen presence. In contrast, we found that PKC inhibition was protective. Three lines of evidence suggest a counteracting link between βE2 and PKC signaling pathways in this neuroprotection model. First, the combined treatment of βE2 and a PKC inhibitor BIM produced a synergistic protection against glutamate toxicity. Second, βE2 inhibited the membrane translocation of PKCε in a manner that correlated with its protection. Finally, βE2 reversed the glutamate-induced increase in the activity of PKCε. These findings support the hypothesis that PKC may be an operative factor in glutamate-induced neuronal cell death (Favaron et al. 1990) and that estrogen suppresses the PKC signaling pathway in an isozyme (PKCε)-specific manner to exert neuroprotection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by AA013864, AG10485 and AG22550. We thank Andrew Wilson for his excellent technical assistance.

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  5. Discussion
  6. Acknowledgements
  7. References
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