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Department of Neurology, Graduate School of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507, Japan; and
* Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyoku, Kyoto 606-8501, Japan
1Correspondence: Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan. E-mail: i53367{at}sakura.kudpc.kyoto-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-estradiol, a stereoisomer without female
hormone activity, also provided an antiapoptotic effect. Therefore, the
antiapoptotic effect is independent of female hormone
activity.—Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Honda, K.,
Nakanishi, M., Akaike, A., Shimohama, S. Mechanisms of
antiapoptotic effects of estrogens in nigral dopaminergic neurons.
Key Words: estrogen receptor ß • AP-1 element • Parkinson’s disease • neuroprotection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cause oxidative stress against
mesencephalic dopaminergic neurons. Oxidative stress can cause neuronal
apoptosis (2
, 3)
, and it has been revealed that radical
induced-apoptosis is involved in the degenerative process of
dopaminergic neurons (4
, 5)
.
Epidemiological studies have indicated that the incidence and
prevalence of Parkinson’s disease is higher in men than in women
(6)
. The ratio of the prevalence in females vs. males is
1:1.36 (7)
~1:3.7 (8)
. Estrogens, which are
female sex steroid hormones, are modulators of apoptosis in the uterine
epithelium (9)
and in breast carcinoma cells
(10)
. It has been discovered recently that estrogens
provide neuroprotection against neuronal degeneration. There have been
several reports that the decline of estrogen is related to the risk of
Alzheimer’s disease in postmenopausal women and that replacement of
estrogen reduces the morbidity of the disease (11
12
13
14)
.
Also, estradiol provides neuroprotection against glutamate- or
radical-induced neurotoxicity in vitro
(15
16
17
18
19)
, including in nigral dopaminergic neurons
(20)
. Recent investigations reported that the
neuroprotection by estradiol is at least partially mediated by
conjugation of estradiol with glutathione (19)
and not
mediated by the estrogen receptor (21)
. Furthermore,
others have reported that activation of extracellular signal-regulated
protein kinase (ERK), an isoform of mitogen-activated kinase, by
estradiol mediates the neuroprotection (22
, 23)
; however,
it is controversial whether or not the ERK activation by estradiol is
mediated by estrogen receptor (22
23
24
25)
.
Recently, a novel subtype (named ER ß) of the estrogen receptor (ER)
was cloned and the classical subtype was renamed ER
(26)
. Estradiol regulates gene transcription in two ways:
through binding to the estrogen response element (ERE) and through the
activator protein-1 (AP-1) enhance element of DNA. Estradiol enhances
gene transcription through the ERE when bound to either ER or ß.
In contrast, it enhances transcription through the AP-1 site when bound
to ER , but suppresses it when bound to ER ß (27)
. In
the central nervous system, the dominant ER subtype is ER ß,
especially in the cerebral cortex, cerebellum, and brainstem. In the
substantia nigra of the mesencephalon, the subtype is exclusively ER
ß (28
, 29)
. In the process of apoptosis, the activator
proteins c-Fos and c-Jun play a pivotal role and may regulate gene
transcription through heteromer formation with the ER (30
, 31)
. Therefore, estradiol may inhibit gene transcription through
the AP-1 site, inhibiting apoptosis in nigral dopaminergic neurons.
L-buthionine-[S,R]-sulfoximine (BSO) is an irreversible specific
inhibitor of 
-glutamylcysteine synthetase (32)
and
causes deprivation of glutathione (33)
. BSO-induced
neurotoxicity can be used as a model of toxicity induced by oxidative
stress in Parkinson’s disease because glutathione is depleted in the
nigral dopaminergic neurons in the disease (34
35
36)
.
Bleomycin sulfate (BLM) is an anti-cancer drug with two molecular
domains: one binds to nuclear DNA and the other acts as a radical
generator (37)
. BSO and BLM have been reported to induce
apoptosis (38
39
40
41)
and can be used as models of oxidative
stress. In the present study, we examined the hypothesis that estrogens
provide neuroprotection against apoptosis induced by experimental
oxidative stress, achieved by exposure to BSO or BLM, in nigral
dopaminergic neurons, and that the antiapoptotic effects are
independent of female hormone activity and mediated by gene
transcriptional regulation through the AP-1 site.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and caspase-3 (Ac-Asp-Met-Gln-Asp-H; Ac-DMQD-CHO)
(43)
were obtained from Peptide Institute Inc. (Osaka,
Japan). Monoclonal anti-tyrosine hydroxylase (TH) antibody was obtained
from Eugene Tech (Ridgefield Park, N.J..), and monoclonal
anti-microtubule associated protein 2 (MAP 2) antibody for
immunocytochemical studies was obtained from Sigma. Anti-c-Jun
NH2-terminal kinase 1 (JNK1),
anti-c-Jun-NH2-terminal kinase 2/3 (JNK 2/3), and
anti-c-Jun antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif.).
Cell culture
Cultures of the rat mesencephalon were performed according to
methods described previously (20
, 44
45
46)
. The ventral
two-thirds of the mesencephalon was dissected from rat embryos on the
16th day of gestation. The dissected regions included dopaminergic
neurons in the substantia nigra and the ventral tegmental area, but not
noradrenergic neurons in the locus ceruleus. Neurons were incubated
with 0.1% trypsin at 37°C for 20 min and incubated with EMEM
containing 40% fetal calf serum (FCS). They were gently triturated and
plated out onto 0.1% polyethyleneimine-coated plastic coverslips at a
density of 1.3 x 105
cells/cm2. The culture medium consisted of EMEM
containing 10% FCS for the first 1 to 4 days in culture and horse
serum (HS) from the 5th day onward. The animals were treated in
accordance with guidelines published in the NIH Guide for the Care and
Use of Laboratory Animals (47)
.
Treatment of the cultures
In a pilot study, 24 h incubation was required to induce
apoptotic morphological changes which were detected by TUNEL and the
nuclear dye Hoechst 33258. Therefore, to investigate BSO- or
BLM-induced neurotoxicity, cultured neurons were exposed to BSO or BLM
for 24 h on the 8th day of culture, incubated in EMEM containing
10% HS for an additional 72 h, and fixed on the 12th day. To
determine the effects of preincubation with estrogens on BSO- or
BLM-induced neurotoxicity, the cultures were preincubated with 10 nM
17- or 17ß-estradiol on the 7th day in culture for 24 h prior
to BSO or BLM exposure. On the 8th day in culture, cells were exposed
to EMEM containing BSO or BLM with 17- or 17ß-estradiol for
24 h. Cells were incubated in medium without drugs for an
additional 72 h and fixed on the 12th day. Control experiments
were similar to treatment, using EMEM containing no drugs. Incubation
with less than 0.1% dimethyl sulfoxide (DMSO) or 0.1% ethanol for
24 h was found to have no effect on neuronal survival rates, and
so 17- and 17ß-estradiol, ICI 182,780, and curcumin were dissolved
in DMSO. Tamoxifen, Ac-WEHD-CHO, Ac-DMQD-CHO, and Yp537 were dissolved
in ethanol. BSO and BLM were dissolved in water.
In a pilot study, concentrations of tamoxifen greater than 1 µM, Yp537 greater than 10 µM, Ac-WEHD-CHO greater than 100 µM and Ac-DMQD-CHO greater than 100 µM were toxic to cultured neurons (data not shown). Therefore, the drug concentrations used were 10–100 nM for tamoxifen, 1 µM for Yp537, and 1–10 µM for Ac-WEHD-CHO or Ac-DMQD-CHO.
Evaluation of neurotoxicity
The number of surviving neurons was determined using
immunostaining as described in our previous studies (20
, 44
45
46)
. Briefly, cultured cells were fixed and incubated with
anti-TH (diluted 1:1000) or anti-MAP2 (diluted 1:400) antibodies for
24 h, with the secondary biotinylated antibody for 1 h, and
with avidin–biotin complex solution (Vectastain) for 1 h.
Finally, the cultures were reacted with diaminobenzidine solution for 6
min. The number of cells stained with anti-TH antibody in 10 to 20
randomly selected fields (x200, total magnification) was taken as the
numbers of surviving dopaminergic neurons and those stained with
anti-MAP2 antibody in 15 randomly selected fields (x400) as that of
total neurons. Counts were made blind to the experimental treatment. In
control cultures, 100 to 200 TH-positive neurons or 100 to 200
MAP2-positive neurons were counted for dopaminergic and total neurons,
respectively. Neurotoxicity was evaluated by the reduction in neuronal
survival rate in each experiment.
Statistical analysis
Statistical analysis was performed by one-way ANOVA and post hoc
multiple comparison by Newman-Keuls’ method when variance in the data
was statistically equivalent. Uniformity of variance was analyzed
statistically by Bartlett’s test. Statistical significance was defined
as P<0.05.
LD50 of the neurotoxicity induced by BSO and BLM
exposure
The dose response curve for BSO toxicity was fitted to the curve
using the following Michaelis Menten’s
equation.
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Evaluation of apoptotic features in cultured cells
To reveal the nuclear morphological changes in cultured neurons,
cells were stained with the nuclear dye Hoechst 33258. On the 8th day,
cells were exposed to BLM and BSO, then fixed with neutral formaldehyde
on the 9th day. They were incubated with 1 mM Hoechst 33258 at room
temperature (RT) for 30 min and washed three times with
phosphate-buffered saline (PBS). Cells were observed under ultraviolet
illumination using fluorescent microscopy.
In situ visualization of DNA fragmentation at the single
cell level was performed by the terminal deoxynucleotidyl transferase
(TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)
method (48)
using the Apoptosis Detection kit (Wako,
Tokyo, Japan). To avoid detachment of fragmented DNA, cells were fixed
with 4% neutral formaldehyde saline immediately after experiments on
the 8th or 9th day in culture and washed twice with PBS, then exposed
to buffer containing sodium citrate (0.1%) and Triton X-100 (0.1%) on
ice for 2 min. Cells were washed for 20 s, incubated with TdT
buffer at 37°C for 10 min, and washed twice with PBS. Endogenous
peroxidase was inactivated with 3%
H2O2 for 5 min at RT. Then
the cells were washed for 10 min and incubated with buffer containing
peroxidase-conjugated antibody at 37°C for 10 min. After washing with
PBS for 10 min, cells were reacted with diaminobenzidine at RT for 5
min and counterstained with methylgreen solution.
Caspase-3 activity
Caspase-3 activity was measured according to methods described
previously (49)
. Briefly, the caspase-3 activity was
detected by the cleavage of the colorimetric caspase-3 substrate,
acetyl-Asp-Glu-Val-Asp-p-nitroanilidine (DEVD-pNA), using an
assay kit, ApoAlert CPP32 (Clontech, Palo Alto, Calif.). Cultured
neurons were harvested using a cell scraper and resuspended in 10 mM
PBS. Cells were collected and incubated with the cell lysis buffer
included in the kit for 10 min on ice and centrifuged at 12,000 rpm for
3 min at 4°C. The supernatant was added to the reaction buffer with
dithiothreitol and the DEVD-p-NA, and incubated for 1 h
at 37°C. Relative caspase-3 activity was measured as optical density
at 405 nm. The relative activity was standardized by a protein
concentration that was determined using the Bio-Rad protein assay kit
(Bio-Rad Laboratories, Hercules, Calif.).
Western blot analysis
Cells were fixed on the 9th day in culture to assay level of
Bcl-2, JNK 1, JNK 2/3 by Western blotting. Cells were washed twice with
cold TBS, harvested using a cell scraper, and lysed in the buffer
containing Tris (40 µM), ß-glycerophosphate (50 mM), EGTA (0.8 mM),
Triton X-100 (2%), phenylmethylsulfonylfluoride(1 mM), aprotinine
(1%), dithiothreitol(2 mM), and vanadate (1 mM) on ice. Lysates were
centrifuged at 150,000 r.p.m. at 4°C for 30 min. The protein was
denatured by boiling at 100°C for 4 min. An aliquot (15 µg as
protein) of the supernatant was loaded onto a sodium dodecyl sulfate
polyacrylamide gel, separated electrophoretically, and transferred to a
polyvinylidene difluoride (PVDF) membrane (Bio-Rad Lab., Inc.). After
the PVDF membrane was incubated with 10 mM TBS with 1.0% Tween 20 and
5% dehydrated skim milk (Difco Lab., W. Molesley, Surrey, U.K.) to
block nonspecific protein binding, the membrane was incubated with
primary antibody [anti-Bcl-2 (1:1000), anti-JNK 1 (1:500), anti-JNK
2/3 (1:500)] or secondary antibody (horseradish peroxidase-linked
antibody, diluted 1:1000). Subsequently, membrane-bound horseradish
peroxidase-labeled antibodies were detected by an enhanced
chemiluminescence detection system (ECL-plus, Amersham) and exposed to
Fuji X-ray film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). After exposure to BSO or BLM or after serum deprivation, cultured
cells were labeled with TUNEL. No TUNEL-positive cells were seen in
control experiment (Fig. 2a
,b
,c
,d
). Nuclear staining with Hoechst 33258 demonstrated that
nuclei were homogeneously stained in the control experiment. After
exposure to BSO or BLM or serum deprivation treatment, nuclear
chromatin was aggregated or condensed (Fig. 2e
,f
,g
,h
).
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Apoptotic neurotoxicity induced by BLM was blocked by Ac-WEHD-CHO
(caspase-1 inhibitor) and Ac-DMQD-CHO (caspase-3 inhibitor); that
induced by BSO was blocked by Ac-DMQD-CHO but not by Ac-WEHD-CHO
(Fig. 3a, b
). Furthermore, BSO- and BLM-induced neurotoxicity was
blocked by coadministration of curcumin, an inhibitor for c-Jun/AP-1
(Fig. 3c, d
). Caspase-3 activity was elevated by BLM
treatment. The elevated caspase activity by BLM was not affected by
curcumin (Fig. 4
).
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Protection by estradiol against BLM- and BSO-induced neuronal death
Preincubation with 1 or 10 nM 17ß-estradiol significantly
protected both types of neurons against toxicity induced by BSO.
17ß-Estradiol preincubation exerted significant neuroprotection
against BLM neurotoxicity in both dopaminergic and nondopaminergic
neurons dose dependently (Fig. 5a, b
). In contrast, 100 nM tamoxifen, an antiestrogen agent,
exaggerated BLM neurotoxicity, and the surviving neuronal number was
decreased from 650 ± 60 to 311 ± 15
cells/cm2 for dopaminergic neurons and from
10,966 ± 537 to 7,841 ± 256 cells/cm2
for nondopaminergic neurons. It also significantly exaggerated the BSO
neurotoxicity to dopaminergic neurons (from 544±54 to 314±21
cells/cm2) and nondopaminergic neurons (from
8,507±652 to 723±224 cells/cm2) (Fig. 5c
). Coadministration with 1 µM ICI 182,780, a pure
antagonist for ERß, antagonized the neuroprotection provided by
17ß-estradiol, but an inhibitor for ERß homodimerization, Yp537 (1
µM), did not block the neuroprotection. ICI 182,780 (1 µM) or Yp537
(1 µM) alone did not exert any detectable effects on neuronal
survival (Fig. 6a
). The neuroprotection provided by 17ß-estradiol was
significant when administered for longer than 2 h; preincubation
for 4 h or longer did not provide additional neuroprotection (Fig. 6b
).
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The activity of caspas-3, which was elevated by BLM, was not suppressed
by preincubation with 10 nM 17ß-estradiol (Fig. 7
). Immunoblotting assay of Bcl-2 revealed that 17ß-estradiol increased
the amount of Bcl-2, but the up-regulation of Bcl-2 required
preincubation for 8 h or longer (Fig. 8
). JNK 1 and JNK 2/3 were elevated by BSO or BLM treatment. The elevated
activity of JNK 1 was suppressed neither by 17ß-estradiol nor by
Ac-DMQD-CHO. In contrast to JNK 1, JNK 2/3 activity was slightly
suppressed by estradiol but was almost completely suppressed by
Ac-DMQD-CHO (Fig. 9
).
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17-Estradiol, a stereoisomer with little biological activity as a
female sex hormone, also significantly protected dopaminergic and
nondopaminergic neurons against apoptosis induced by BSO (Fig. 10
). Preincubation with either 17- or 17ß-estradiol significantly
reduced the number of TUNEL-labeled cells after BSO exposure (the
number of TUNEL-positive cells induced by BSO was decreased from
2,223±271 cells/cm2 to 185±46
cells/cm2 and 46±26
cells/cm2 by 17- and 17ß-estradiol,
respectively) (Fig. 10)
. Table 1
presents the neuroprotection of 17- and 17ß-estradiol against
apoptosis induced by various agents. Preincubation with either 17-
or 17ß-estradiol provided significant neuroprotection against
apoptosis induced by exposure to BLM and by serum deprivation, as well
as that induced by BSO. In contrast to preincubation with estradiol,
preincubation with other steroids, such as corticosterone,
testosterone, and cholesterol, had no effect on neuronal apoptosis
induced by BLM exposure (Table 2
).
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As shown in Fig. 3a, b
, apoptotic neurotoxicity induced by
BLM was blocked by Ac-WEHD-CHO (caspase-1 inhibitor) and Ac-DMQD-CHO
(caspase-3 inhibitor), and that induced by BSO was blocked by
Ac-DMQD-CHO but not by Ac-WEHD-CHO. In contrast, neuronal death induced
by serum deprivation was not blocked by either inhibitor (Fig. 11
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, DNA damage in necrotic
cells may cause nonspecific staining by TUNEL (50
51
52)
. In
addition to DNA cleavage, nuclear staining using Hoechst 33258 showed
the aggregation and condensation of nuclear chromatin. Furthermore,
neurotoxicity by BLM and BSO was significantly blocked by a caspase-3
inhibitor. Taken together, the neuronal death induced by BSO and BLM is
taken as apoptosis according to the morphological and biochemical
criteria of apoptotic cell death (53)
.
Although BSO was toxic to both dopaminergic and nondopaminergic
neurons, the LD50 was higher for dopaminergic
neurons than for nondopaminergic neurons, showing that dopaminergic
neurons were relatively resistant to the toxicity. The reason why
dopaminergic neurons were less vulnerable to BSO is unknown. However,
dopaminergic neurons are resistant to another oxygen radical, nitric
oxide (44
, 45)
, and they may have a great ability to
scavenge radicals (34)
. Therefore, they may be relatively
resistant in general to toxicity induced by reactive oxygen species.
BSO-induced apoptosis is thought to be mediated by an increase in
intracellular H2O2 because
it deprives the cells of glutathione and suppresses glutathione
peroxidase. Apoptosis induced by reactive oxygen species such as
H2O2 or
O2- is mediated by caspase-1
(54
, 55)
or caspase-3 (56)
. In the present
study, BSO-induced apoptosis was not blocked by Ac-WEHD-CHO, a
caspase-1 inhibitor, but by Ac-DMQD-CHO, a caspase-3 inhibitor.
Therefore, BSO-induced neurotoxicity was mediated by caspase-3
activation. BLM-induced apoptosis is mediated by the activation of
caspases-1 and -3, because it was blocked by caspase-1 and caspase-3
inhibitors. Caspase-3 is thought to be activated downstream of
caspase-1 (57)
. Therefore, the pathway of BLM-induced
neuronal apoptosis involves activation of caspase-1, followed by
activation of caspase-3.
Apoptosis induced by anti-cancer drugs and oxygen radicals such as
hydrogen peroxide requires the activation of AP-1 and transcriptional
activation through the AP-1 element (30
, 31
, 58
59
60
61
62)
.
Also, in the present study apoptosis by BSO or BLM was blocked by
curcumin, a pharmacological inhibitor for c-Jun/AP-1 proteins. c-Jun
was activated by JNK, a family of mitogen activator protein kinases,
and JNK 2/3 was suppressed by a caspase-3 inhibitor, as shown in Fig. 9
. Furthermore, activation of caspase-3 by BLM was not blocked by
curcumin, as shown in Fig. 4
. Therefore, the c-Jun/AP-1 proteins were
activated downstream of caspase-3. These data are consistent with a
recent report by Srivastava (63)
.Therefore, apoptosis by
BSO or BLM is possibly mediated by the AP-1 activation downstream of
caspase-3.
Neuroprotection by estrogens
17ß-Estradiol provides neuroprotection against the apoptosis
induced by these agents. The antiapoptotic neuroprotection was not
attributable to a general steroid structure because other steroids
provided no protection against apoptosis. A possible mechanism is the
antioxidant property of estradiol. Steroids with hydroxyl group in C3
position of the A ring provided an antioxidant property
(17)
. In our previous report, 17ß-estradiol suppressed
intracellular oxygen radicals and provided neuroprotection against
oxidative stress; however, antioxidant property requires a much higher
concentration of estradiol (10–100 µM) (20)
.
Furthermore, antiapoptotic neuroprotection was blocked by ICI 182,780,
which has hydroxyl group in C3. Therefore, it is unlikely that the
antiapoptotic effect of estradiol is due to the antioxidant property of
estradiol.
Recently, it has been reported that estradiol causes up-regulation of
Bcl-2 (64
65
66)
, which suppresses cytochrome c
release and the ensuing caspase-3 activation. In our mesencephalic
culture, estradiol caused up-regulation of Bcl-2, but it required
estradiol treatment for longer than 8 h (Fig. 8)
. As shown in Fig. 6
, preincubation for only 2 h provided significant antiapoptotic
neuroprotection. It is unlikely that up-regulation of Bcl-2 plays a
pivotal role in antiapoptotic protection because of different time
dependency. Furthermore, we recently reported that glial cell
line-derived neurotrophic factor (GDNF) causes up-regulation of Bcl-2
in the mesencephalic neurons and provides neuroprotection against BSO-
and BLM-induced apoptosis, but GDNF does not protect neurons from
apoptosis by serum deprivation (49)
. In contrast to GDNF,
estradiol provided neuroprotection from apoptosis induced by serum
deprivation treatment. Therefore, antiapoptotic protection by estradiol
in the present study includes other mechanisms besides Bcl-2
up-regulation.
The neuroprotection provided by 17ß-estradiol was thought to be
mediated by the ER because it was antagonized by ICI 182,780, a pure
antagonist for ER ß. 17ß-Estradiol has affinity for both
subtypes of ER (29)
, ER and ER ß, but the
neuroprotection of nigral dopaminergic neurons is probably mediated by
ER ß, because it has been revealed that the ER subtype in the
substantia nigra of the mesencephalon is exclusively ER ß (28
, 29)
. Gene expression as a result of ligand-bound ER includes
both primary and secondary responses, with the former directly
regulated by the ER and the latter regulated by products of the primary
response. The primary response to ligand-bound ER is mediated in two
ways: through the ERE and through the AP-1 element. ER ß activated by
17ß-estradiol binds to DNA as a homodimer and enhances gene
transcription through the ERE. However, it also suppresses gene
transcription from the AP-1 element by coupling with he AP-1 proteins
c-Fos and c-Jun (27)
. Tamoxifen acts as a pure antagonist
for ER ß (67)
and, when bound to ER ß, regulates
transcription through either the ERE or the AP-1 element in a reverse
manner: it suppresses transcription through ERE and enhances it through
AP-1 site (27)
. In this study, it exaggerated the
apoptotic toxicity induced by BSO and BLM. Therefore, the antiapoptotic
effect of 17ß-estradiol could be mediated by the activation of
antiapoptotic genes through the ERE or by the suppression of
proapoptotic genes through the AP-1 enhancer element.
The neuroprotection provided by 17ß-estradiol in the present study
was not antagonized by Yp537. Yp537 is a synthesized 12-amino acid
phosphotyrosyl peptide containing a selected sequence surrounding
tyrosine-537, a constitutively phosphorylated site, on the estrogen
receptor (68)
. It blocks the homodimer formation of the ER
and effects of the pathway regulated by the ERE, but not the effects of
the activation of the AP-1 site, because ligand-bound ER attaches to
the AP-1 consensus element by making a heteromer with he AP-1 proteins
Fos and Jun. These findings suggest that the 17ß-estradiol-ER ß
complex inhibits the transcription of certain proapoptotic genes from
the AP-1 element that suppress apoptosis. Therefore, the antiapoptotic
effect by estradiol is thought to be mediated by suppression of
proapoptotic genes through the AP-1, but not ERE.
Serum deprivation for 48 h caused significant neurotoxicity
resulting in a large number of TUNEL-positive cells. Nuclear staining
using Hoechst 33258 showed that there was nuclear aggregation after
serum deprivation treatment. Therefore, neuronal death caused by serum
deprivation was accompanied by the nuclear morphological features
characteristic of apoptosis and by DNA cleavage, and was thought to be
apoptosis. In contrast to apoptosis induced by BSO or BLM, that induced
by serum deprivation was not blocked by either Ac-WEHD-CHO or
Ac-DMQD-CHO. These data are consistent with a recent report that
apoptosis induced by potassium deprivation does not require activation
of caspase-3 (69)
and indicate that serum deprivation
caused apoptosis at least partially by a direct pathway to cascades
downstream from the activation of caspase-3, such as activation of the
AP-1 proteins or endonuclease in the apoptotic process
(70)
. Estradiol provided antiapoptotic protection against
serum deprivation treatment. Furthermore, estradiol had no effect on
caspase-3 or JNK activation. Therefore, the key proapoptotic gene
products inhibited by estradiol are most likely downstream from the
activation of caspase-3 and JNK (Fig. 12
). In addition to 17ß-estradiol, 17-estradiol also provided
significant antiapoptotic neuroprotection, consistent with a recent
study by Green et al. (71)
using a neuroblastoma cell
line. However, the estradiol-induced neuroprotection reported by Green
et al. might be mediated by a different mechanism from that in nigral
neurons because the neuroprotection in their report was not antagonized
by tamoxifen, in contrast to our results. Although it is not known
which subtype of ER mediates neuroprotection in the neuroblastoma cell
line, the different response to tamoxifen may be related to the
subtypes of ER mediating neuroprotection, since tamoxifen does not
antagonize transcription regulation through AP-1 when mediated by ER
(27
, 67)
.
|
Compared to 17ß-estradiol, 17-estradiol has few of the biological
effects of female sex steroid hormones. A ligand binding study showed
that 17-estradiol has affinity for ER ß (relative binding affinity
of 17- and 17ß-estradiol is 0.11:1) (29)
. Therefore,
17-estradiol can activate ER ß and cause the same primary response
as that of 17ß-estradiol, at least when an appropriate dose is
applied. The antiapoptotic protection by estradiol is probably the
results of a primary response initiated by estradiol because
preincubation for only 2 h provided significant antiapoptotic
protection in the present study. The negative biological estrogenic
effects of 17-estradiol may be a result not of the difference in
affinity, but rather of the difference in secondary response induced by
17- and 17ß-estradiol, since the 17- isomer is bound to ER
and ER ß for a shorter duration than the 17ß-isomer
(72)
. 17-Estradiol, or its analog, may be a candidate
for antiapoptotic therapy in neurodegenerative diseases affecting
neurons expressing ER ß, such as Parkinson’s disease, because it has
few of the biological effects associated with female sex
hormones.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B is increased in dopaminergic neurons of patients with Parkinson disease. Proc. Natl. Acad. Sci. USA 94,7531-7536 and ERß at AP1 sites. Science 277,1508-1510 and -ß mRNA in the rat central nervous system. J. Comp. Neurol. 388,507-525[Medline]
and ß. Endocrinology 138,863-870 B and AP-1. Mol Cell 1,543-551[Medline]
B, is a mediator for oxidant-initiated apoptosis in glomerular mesangial cells. Biochem. Biophys. Res. Commun. 240,496-501[Medline]
and estrogen receptor ß to partial estrogen agonists/antagonists. Mol. Pharmacol. 54,105-112-Estradiol exerts neuroprotective effects on SK-N-SH cells. J. Neurosci. 17,511-515This article has been cited by other articles:
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) and nondopaminergic (
) neurons. a)
Neurotoxicity induced by 3 µM BSO was not blocked by the caspase-1
inhibitor Ac-WEHD-CHO, but was significantly blocked by an inhibitor of
caspase-3, Ac-DMQD-CHO. *P<0.001 compared to control,
P<0.001 compared to BSO. n = 3
coverslips/experiment. Error bars represent SEM.
b) Neurotoxicity induced by exposure to 30 µM BLM was
significantly blocked by preincubation with either Ac-WEHD-CHO (a
caspase-1 inhibitor) or Ac-DMQD-CHO (a caspase-3 inhibitor) in both
dopaminergic and nondopaminergic neurons. *P<0.001
compared to control, 
