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NEUROENDOCRINOLOGY |
Department of Molecular Pharmacology and Toxicology and the Program in Neuroscience, University of Southern California, Pharmaceutical Sciences Center, Los Angeles, California 90033
Address all correspondence and requests for reprints to: Roberta Diaz Brinton, Ph.D., Molecular Pharmacology and Toxicology, University of Southern California, Pharmaceutical Sciences Center, 1985 Zonal Avenue, Los Angeles, California 90089. E-mail: rbrinton{at}hsc.usc.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is becoming increasingly apparent that HRT is beneficial in the central nervous system, in part, because of the neurological effects of estrogens, including protection against neurologic insults, reduced risk of Alzheimer’s disease (AD), and improved cognitive function (8, 9). In vitro models for these effects have shown that estrogen enhances neuronal survival resulting from oxidative stress, excitotoxic insults, and ß-amyloid (10, 11). Although all the mechanisms underlying these effects remain to be identified, recent studies have shown that the tyrosine kinase/MAPK signal transduction cascade may be involved in estrogen-mediated neuroprotection. Estrogen rapidly activated tyrosine kinase and MAPK activity (12, 13, 14), and the neuroprotective effect of estrogen against glutamate toxicity was blocked by inhibitors of tyrosine kinase and MAPK (15). The MAPK pathway is thought to play an important role in the actions of neurotrophins, and its activation could lead to increased expression of antiapoptotic genes. One such antiapoptotic gene, bcl-2, is involved in survival of nerve growth factor (NGF)-dependent sensory neurons (16) and inhibits death in response to glutamate toxicity (17). Additionally, Bcl-2 expression is increased in response to estrogen treatment of neuronal cells (18, 19, 20).
Very little research on hormonal modulation of neuronal survival has studied the effects of progestins only or progestins in conjunction with estrogen. It is currently unknown what effect different progestins have on estrogen-mediated modulation of MAPK activation and Bcl-2 expression in neuronal cells. It is not clear whether progestins are neuroprotective or not, nor whether they impact the neuroprotective effects of estrogen. Previous studies reported no neuroprotective effect of progestins (21), whereas others have seen positive effects of progestin on neuronal survival (22, 23). Direct comparison between these studies is difficult, because each differed in the neuroprotective model studied and in the progestin used.
The purpose of this study was to determine the effects of three widely clinically used progestins [progesterone, 19-norprogesterone, and medroxyprogesterone acetate (MPA)] on a well-established model of estrogen-induced neuroprotection. Results of the present study demonstrate that progesterone and 19-norprogesterone were neuroprotective, alone or when administered in conjunction with 17ß-E2, whereas MPA was not. Not only was MPA an ineffective neuroprotectant, it blocked estrogen-induced neuroprotection when coadministered. Although all three progestins tested activated MAPK, only the neuroprotective progesterone and 19-norprogesterone increased the expression of Bcl-2. These data point out the necessity of examining the composition of hormones used in HRT formulations, especially with regard to which progestin is included. This work belies a common underlying theme in endocrinology, that although structurally different ligands behave the same in some systems, they do not necessarily do so in all systems.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neuronal culture
Primary cultures of dissociated hippocampal neurons were
performed as previously described (24). Briefly,
hippocampi were dissected from the brains of E18 rat fetuses, treated
with 0.02% trypsin in HBSS (50 mM KCl, 3 mM
HH2PO4, 80 mM
NaCl2, 0.9 mM
Na2HPO4·7H2O,
10 mM dextrose, 0.3 M HEPES), for 5 min
at 37 C, and dissociated by repeated passage through a series of
fire-polished constricted Pasteur pipettes; and
106 cells/ml were plated on PEI-coated 96-well
plates for toxicity experiments or 6-well plates for immunoblots. Nerve
cells were grown in Neurobasal medium (Life Technologies, Inc., Rockville, MD) supplemented with 10 mM
NaHCO3, 5 U/ml penicillin, 5 mg/ml streptomycin,
and B27 supplement (Life Technologies, Inc.). Cultures
were maintained at 37 C in a humidified 5% CO2
atmosphere, and all experiments were performed after 12 d in
culture.
Glutamate toxicity
Forty-eight hours before glutamate exposure, cultures were
placed in fresh Neurobasal medium supplemented with B27, 10
mM NaHCO3, 5 U/ml penicillin, and 5
mg/ml streptomycin, with the steroids as indicated. 17ß-E2
(Sigma), progesterone (Sigma, St.
Louis, MO), 19-norprogesterone (Sigma), and MPA
(Pharmacia & Upjohn, Inc., Peapack, NJ) were all
dissolved in ethanol, to a stock solution of 1 mg/ml; and further
dilution was in culture medium so that the final concentration of
ethanol was less than 0.01%. Glutamate exposure was performed for 5
min at 37 C in buffer containing 2 mM KCl, 1 mM
MgSO4, 2.5 mM
CaCl2, 1 mM
NaHPO4, 4.2 mM
NaHCO3, 12.5 mM HEPES, 10
mM glucose, 0.1 M NaCl, and 100
µM L-glutamic acid. Cultures were then washed
and returned to fresh Neurobasal medium.
Evaluation of plasma membrane damage
Overall neuronal injury was assessed 24 h after the start
of the exposure by quantitative measurement of lactate
dehydrogenase (LDH) in the bathing medium, an index that is
proportional to the total number of neurons damaged by excitotoxic
exposure (25). LDH activity was measured using the
Cytotoxicity Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN) and quantitated by
measuring the absorbance at 490 nm. Data are normalized against the
amount of LDH activity released from vehicle-treated control cultures
receiving no glutamate. Data are presented as the mean ±
SEM for at least three independent experiments.
MAPK phosphorylation
Hormones were added to the cultures, as indicated, for 30 min;
and the cells were lysed by incubation in ice-cold lysis buffer
(0.005% SDS, 0.1% Igepal, 0.2 mM sodium orthovanadate,
and 0.2 mM phenylmethylsulfonylfluoride in PBS) for 30 min
at 4 C. Cell lysates were cleared by centrifugation at 12,000 x
g for 10 min, and the concentration of protein in the
supernatant was determined by the BCA Protein Assay
(Sigma). Twenty micrograms of total protein from
whole-cell lysates were separated under reducing and denaturing
conditions by 12% SDS-PAGE and were electrotransferred to
polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Nonspecific binding sites were
blocked with 5% skim milk in PBS containing 0.05% Tween-20
(PBS-Tween). An antibody recognizing the dual threonine and tyrosine
phosphorylation sequence from MAPK necessary for activation of the
enzyme (antiactive MAPK; Promega Corp., Madison,
WI) was used to evaluate ERK1/ERK2 phosphorylation. The membranes were
incubated with antiactive MAPK antibody (1:2,000 in PBS-Tween/1% horse
serum) or total ERK2 antibody (C-14) (1:5,000 in PBS-Tween/1% horse
serum; Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) for 1 h. Membranes were then incubated in horseradish
peroxidase (HRP)-conjugated horse antimouse IgG (1:10,000), and results
were visualized by the TMB Peroxidase Substrate Kit (Vector Laboratories, Inc., Burlingame, CA). Relative amounts of
phospho-ERK and total ERK2 were quantitated by optical density analysis
using Scion Image software (Scion, Frederick, MD). The level of
phospho-ERK was normalized, with respect to total ERK2. So as to avoid
interassay variations, the values obtained were also normalized with
the value measured for the vehicle-treated control cultures in each
experiment. Data are presented as the mean ±
SEM for at least three independent
experiments.
Bcl-2 expression
Hormones were added to the cultures, as indicated, for 24 h
before collection of whole-cell lysates and SDS-PAGE as above. Blocked
membranes were incubated with anti-Bcl-2 antibody (1:250 in
PBS-Tween/1% horse serum; Zymed Laboratories, Inc., San Francisco, CA) for 1 h. Membranes were
then incubated in horseradish peroxidase (HRP)-conjugated horse
antimouse IgG (1:10,000), and results were visualized by the TMB
Peroxidase Substrate Kit (Vector Laboratories, Inc.).
Relative levels of Bcl-2 were quantitated by optical density analysis
using Scion Image software. So as to avoid interassay variations, the
values obtained were also normalized with the value measured for the
vehicle-treated control cultures in each experiment. Data are presented
as the mean ± SEM for at least three independent
experiments.
Statistics
Statistically significant differences between groups were
determined by an ANOVA followed by a Newman-Keuls post hoc
analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; P < 0.05, compared
with control; n = 4). As expected, 17ß-E2 significantly reduced
the amount of LDH released into the media 24 h after exposure to
100 µM glutamate (Fig. 1). The magnitude of
neuroprotection we report is effectively similar to that previously
reported by other groups using primary cortical cultures, when the data
are analyzed in the same manner (15). Maximum
neuroprotection, of approximately 18% reduction in LDH release,
occurred at 10 ng/ml 17ß-E2 (Fig. 1; P < 0.05,
compared with glutamate alone; n = 4) and was thus used in
subsequent experiments.
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). Maximal neuroprotection, of
approximately 22% reduction in LDH release, was observed at 10 ng/ml
(Fig. 2A; P < 0.05, compared with glutamate alone;
n = 4). 19-Norprogesterone significantly reduced the amount of LDH
released into the media 24 h after a 5-min
100-µM glutamate exposure (Fig. 2B). Maximal
neuroprotection, of approximately 21% reduction in LDH release, was
observed at 10 ng/ml (Fig. 2B; P < 0.05, compared with
glutamate alone; n = 4).
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).
Effect of coadministration of 17ß-E2 and progestins on
glutamate-induced neurotoxicity
Increasingly, progestins are being added to HRT, necessitating the
study of the impact of progestin coadministration on the
neuroprotective effects of estrogen. A 48-h pretreatment of 10 ng/ml
17ß-E2, before a 5-min 100-µM glutamate exposure,
decreased LDH release approximately 17%, compared with vehicle-treated
cultures exposed to glutamate (Fig. 3;
P < 0.05, compared with glutamate alone; n = 4).
A slightly larger magnitude of neuroprotection was observed when
progesterone (
24% of glutamate alone LDH release) (Fig. 3A;
P < 0.05, compared with glutamate alone; n = 4)
or 19-norprogesterone (23% of glutamate alone LDH release) (Fig. 3B; P < 0.05, compared with glutamate alone; n =
4) was coadministered along with the E2. In contrast to the other
progestins tested, no significant decrease in LDH release occurred when
MPA was coadministered with 17ß-E2, compared with vehicle-treated
cultures exposed to glutamate (Fig. 3C; n = 4). MPA-induced
antagonism of 17ß-E2 was greatest at 10 ng/ml (Fig. 3C; n = 4).
Subsequent experiments used 10 ng/ml of all three progestins.
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Treatment with 17ß-E2 (10 ng/ml for 30 min) resulted in approximately
60% increase in phospho-ERK2 immunoreactivity (Fig. 4), consistent with previous reports
(14, 15). In parallel to effects on neuroprotection,
progesterone (10 ng/ml for 30 min) or 19-norprogesterone (10 ng/ml for
30 min) treatment increased phospho-ERK2 immunoreactivity by
approximately 123% (Fig. 4A) and approximately 50% (Fig. 4B),
respectively. Coadministration of progesterone or 19-norprogesterone
with 17ß-E2 increased phospho-ERK2 immunoreactivity by approximately
100% (Fig. 4A) and approximately 75% (Fig. 4B), respectively,
compared with vehicle-treated cultures. In contrast to its effects on
neuroprotection, treatment with MPA (10 ng/ml for 30 min) increased
phospho-ERK2 immunoreactivity approximately 60% (Fig. 4C) and did not
antagonize E2’s effect on MAPK activation when coadministered (Fig. 4C).
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). Progesterone (10 ng/ml for 24 h)
or 19-norprogesterone (10 ng/ml for 24 h) treatment resulted in
increases in Bcl-2 expression of approximately 3-fold (Fig. 5A) and
approximately 2.6-fold (Fig. 5B), respectively. In contrast, MPA (10
ng/ml for 24 h) did not increase Bcl-2 immunoreactivity, compared
with vehicle-treated control cultures (Fig. 5C).
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) and approximately 3.3-fold (Fig. 5C),
respectively, compared with vehicle-treated control cultures.
Consistent with the results on neuroprotection, coadministration of MPA
effectively blocked E2-induced increase in Bcl-2 immunoreactivity
(Fig. 5C). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well documented that women have a greater risk than men to develop AD (26). In addition, females sustain lower mortality and less neuronal damage after cerebral ischemia than males (27). This protection against ischemic brain injury and its related mortality is greatly diminished after menopause or ovariectomy (28). This suggests that the sex-dependent increased risk for neurologic damage is attributable, in part, to the deprivation of ovarian hormones in postmenopausal women.
Although estrogen has been shown to have neuroprotective effects in numerous experimental models, the clinical data on the protective effects of estrogen replacement therapy (ERT) in women at risk for AD is less clear. Epidemiological studies have demonstrated that women who receive ERT are less likely to develop AD or develop AD with a later onset(27, 29, 30, 31). However, some studies have reported no effect of hormones on the development of AD (32).
Vongher and Frye (22) found that rats treated with progesterone, alone or in conjunction with estrogen, before neural insults, had improved cognitive behavior. Other groups have reported no effect of progestin on neuroprotection (21). Here, we show that progesterone, as well as 19-norprogesterone, protects hippocampal neurons against glutamate excitotoxicity. This effect is seen when progesterone is administered alone or in conjunction with 17ß-E2. In contrast, MPA is ineffective when administered alone. When administered in conjunction with E2, it antagonizes the neuroprotective effect of E2. This fits with the clinical data indicating negative effects of synthetic progestins in the treatment of women with AD. Two recent clinical studies found beneficial effects of long-term ERT on cognitive function, but these improvements were abrogated by the addition of MPA (33, 34).
Although ER and PR generally function as transcription factors, a large body of evidence now documents rapid effects of estrogen that are not in accordance with classical genomic mechanisms of hormone action (13, 35). In particular, attention has been focused on the ability of estrogen to activate MAPK pathways, including p42/44 (ERK1/ERK2) (12, 13, 14). Furthermore, estrogen-mediated neuroprotection is thought to involve MAPK activation, because inhibitors of tyrosine kinase and MAPK blocked the neuroprotective effect of estrogen against glutamate toxicity (14, 15, 36). Our results show that neuroprotective concentrations of 17ß-E2, progesterone, and 19-norprogesterone cause rapid activation of MAPK. Surprisingly, MPA, which blocked estrogen- induced neuroprotection, also activated MAPK when administered alone or in conjunction with 17ß-E2. Although previous studies showed that blocking MAPK activation inhibited estrogen-induced neuroprotection, indicating a necessity for MAPK activation, our results imply that MAPK activation is necessary, but not sufficient, for protection. Alternatively, different upstream activators may produce different MAPK-induced effects, as shown for fibroblast growth factor, NGF, and epithelial growth factor effects on PC12 cells. Fibroblast growth factor and NGF induce differentiation, whereas epithelial growth factor acts as a mitogen, even though all three activate MAPK (37). It is possible that a similar mechanism accounts for the different effects of progestins that all activate MAPK.
An alternative proposed mechanism of estrogen-mediated neuroprotection is up-regulation of Bcl-2 expression. The protein Bcl-2, initially identified in hematopoietic tissues, has more recently been found to be ubiquitous. Its main function seems to be to override apoptosis. Bcl-2 has been shown to inhibit neuronal death caused by multiple insults, including growth factor withdrawal, free radicals, and glutamate excitotoxicity (17, 38, 39). Estrogen has been shown to increase Bcl-2 expression in many tissues types, including primary neuronal cultures and neural cell lines (18, 19, 20). Here, we confirm that there is an E2-induced increase in Bcl-2 expression in primary neurons. Progesterone or 19-norprogesterone, administered alone or in conjunction with 17ß-E2, resulted in increased Bcl-2 expression as well. Consistent with MPA inhibition or blockade E2-induced neuroprotection, MPA blocked the estrogen-induced increase in Bcl-2 expression. This further implicates the role of Bcl-2 in ovarian hormone-mediated neuroprotection.
A previous study on breast cancer cells showed a negative regulatory effect of progestin on Bcl-2 expression (40). Although the negative regulatory effect may reflect a difference in response between breast cancer cells and neurons, the progestin used was the synthetic pregnane progestin, OR2058. This synthetic progestin may act more like the synthetic progestin MPA than like natural progesterone. This stresses further the need to study structurally different progestins for differing effects.
Considered together, these results are consistent with the emerging clinical data indicating divergent actions of different progestins. Studies comparing the associations between unopposed estrogen use and combined estrogen- progestin therapy, including the Postmenopausal Estrogen/Progestin Interventions trial, have shown that MPA, but not micronized progesterone, blunted the beneficial association between estrogen and high-density lipoprotein cholesterol (41, 42). Likewise, even though progesterone can protect against increased risk of breast cancer associated with unopposed estrogen (6), the risk of breast cancer was higher with MPA plus estrogen than with estrogen alone (7). The Kame project reported that the beneficial association between estrogen and cognitive change was opposed by the addition of MPA (34). Here, we show that progesterone and 19-norprogesterone plus E2 protected, but MPA plus E2 failed to protect, against neurotoxic insults. These findings may have important implications for the effective use of HRT in the maintenance of neuronal function during menopause and aging and for protection against neurodegenerative diseases such as AD.
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Footnotes |
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Abbreviations: AD, Alzheimer’s disease; ERT, estrogen replacement therapy; HRT, hormone replacement therapy; LDH, lactate dehydrogenase; MPA, medroxyprogesterone acetate; NGF, nerve growth factor; PBS-Tween, PBS containing 0.05% Tween-20.
Received March 7, 2001.
Accepted for publication September 13, 2001.
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