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Endocrine Unit (S.B., P.L., E.F., M.S., A.P.) and Clinical Biochemistry Unit (S.G.), Department of Clinical Physiopathology, Center for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative, and Neoplastic Disorders, University of Florence; and Department of Anatomy, Histology, and Forensic Medicine (G.B.V.), University of Florence, 50139 Florence, Italy
Address all correspondence and requests for reprints to: Alessandro Peri, M.D., Ph.D., Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy. E-mail: a.peri{at}dfc.unifi.it.
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Abstract |
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and ERß. We found that 17ß-estradiol (17ßE2) and the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen exerted neuroprotective effects, which were independent of cell proliferation, by increasing resistance against ß-amyloid-induced toxicity, with the exception of the highest concentrations of raloxifene (10 and 100 nM). In addition, 17ßE2 exposure protected from oxidative stress, reduced apoptosis, and increased the expression of the catalytic subunit of telomerase. Furthermore, we evaluated by quantitative real-time RT-PCR whether estrogen/SERMs modulate the expression of the recently discovered seladin-1 (selective AD indicator-1) gene, which exerts neuroprotective effects and is down-regulated in AD-vulnerable brain regions. 17ßE2 (100 pM to 100 nM) and SERMs (1 nM) significantly increased the amount of seladin-1 mRNA. Conversely, 10 and 100 nM raloxifene reduced the expression of seladin-1. The effect of estrogen appears mainly mediated by ER because the selective ER agonist propylpyrazole-triol determined a much greater increase of seladin-1 expression than the ERß agonist diarylpropionitrile. Our results add new evidence, using human neuronal cells, for a beneficial effect of estrogen in preventing neurodegenerative diseases and suggest for the first time that seladin-1 may mediate this effect. |
Introduction |
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and ERß have been found, for instance, in the neocortex and hippocampus, two brain areas highly involved in neurodegenerative disorders, such as Alzheimer’s disease (AD) (2). AD, which is the most prevalent form of late-life mental failure in humans, is characterized by a progressive impairment of cognitive functions, such as memory and language. The histopathological hallmark of AD is represented by the accumulation of extracellular ß-amyloid plaques and intracellular neurofibrillary tangles, which are responsible for a complex inflammatory response leading to neuronal degeneration and cell death (reviewed in Ref.3). Because there is still no reliable way of predicting the onset of the disease and curing it, AD constitutes a profound health, social, and economic burden. The above-mentioned in vitro studies claim a favorable estrogen effect in protecting from AD. AD is more common in women, and decreased estrogen levels after menopause are a risk factor for the disease (4). Despite the lack of general consensus, many studies indicate that estrogen treatment may decrease the risk or delay the onset of AD in postmenopausal women (reviewed in Ref.5).
Recently a novel gene, named seladin-1 (for selective AD indicator-1), has been isolated and found to be down-regulated in brain regions affected by AD (6). Seladin-1 expression has also been demonstrated in different organs other than the brain (i.e. lung, adrenal gland, testis, ovary, prostate, liver, stomach) (6). Remarkably, overexpression of seladin-1 in neuroglioma H4 cells conferred protection against ß-amyloid-mediated toxicity and from oxidative stress. In addition, seladin-1 effectively inhibited caspase-3 activity, a key mediator of apoptosis, and protected from apoptotic death (6). This gene has marked sequence homology to the Diminuto/Dwarf1 gene, described in plants (i.e. Arabidopsis thaliana) and Caenorhabditis elegans (7). In plants Diminuto/Dwarf1 is required for the synthesis of brassinosteroids, which are plant sterols essential for normal growth and development (8, 9). A subsequent study demonstrated that seladin-1 is the gene encoding 3ß-hydroxysterol 
24-reductase (10), which catalyzes the reduction of the 24 double bond in desmosterol to produce cholesterol. Mutations of this gene have been found in desmosterolosis, a rare severe multiple-congenital-anomaly syndrome, including developmental and growth retardation (10). There are no data, so far, to show whether estrogen modulates the expression of seladin-1.
We established, cloned, and propagated previously GnRH-secreting neuroblast long-term cell cultures from human fetal olfactory epithelium (11, 12). These cells [fetal neuroepithelial cells (FNC)] show unique features because they synthesize both neuronal proteins and olfactory markers and respond to odorant stimuli, suggesting their origin from the stem cell compartment that generates mature olfactory receptor neurons. In addition, FNC express both ER and ERß (12). Thus, they represent a good, and most importantly human, in vitro model that can be of help in providing additional information on the role of estrogen in neurons. We determined the effect of 17ß-estradiol (17ßE2) and the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen on proliferation rate and resistance against ß-amyloid-induced toxicity. In addition, we evaluated the effect of 17ßE2 on apoptosis, resistance against oxidative stress, and expression of the catalytic subunit of telomerase i.e. human telomerase reverse transcriptase (hTERT). Finally, we investigated for the first time whether 17ßE2, raloxifene, tamoxifen, and ER or ERß agonists modulate the expression of seladin-1.
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Materials and Methods |
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Media and sera for cell cultures were purchased from Life Technologies (Grand Island, NY), and tissue plasticware was obtained from Falcon (Oxnard, CA). 3H-thymidine (6.7 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). 17ßE2 and tamoxifen were purchased from Sigma (Milan, Italy) and dissolved in absolute ethanol to a final concentration of 0.1 mM. Raloxifene was obtained from Eli Lilly (Indianapolis, IN) and dissolved in dimethylformamide to a final concentration of 0.1 mM. Propylpyrazole-triol (PPT), a well-characterized agonist of ER, and diarylpropionitrile (DPN), an ERß selective agonist (13), were obtained from Tocris Cookson Ltd. (Bristol, UK) and were dissolved in absolute ethanol to a final concentration of 100 mM. Human ß-amyloid peptide was obtained from Calbiochem Corp. (San Diego, CA), solubilized in 5% acetic acid, and stored at –20 C.
Cell cultures
FNC were isolated from human fetal olfactory neuroepithelium, as described previously (11). Briefly, the mucosa lining the upper nasal cavity and septum was removed from human fetuses (8–12 wk) and dissected after spontaneous or therapeutic absorption. Tissue fragments were washed, digested, and dispersed by pipetting. Cell suspensions were washed, and erythrocytes and debris were removed by centrifugation. Olfactory cells were cloned, and long-term cell cultures were established and propagated in Coon’s modified Ham’s F12, supplemented with 10% fetal calf serum (FCS) and antibiotics (growth medium) as described previously (11). FNC stained positively for neuronal and olfactory markers, such as neuron-specific enolase and vimentin, which target maturing olfactory receptor neurons, and neural cell adhesion molecule and olfactory marker protein, which appear at a relatively later stage during differentiation (11). The B4 clone, which showed the highest levels of expression of neuronal and olfactory markers, was used in this study. In addition, the B4 clone stained positively for both the ER and ERß (12). In the experiments in which the cells were treated with 17ßE2, raloxifene, or tamoxifen, the growth medium was without phenol red and supplemented with 1% charcoal-stripped FCS.
Cell proliferation
FNC-B4 cell growth was evaluated by both 3H-thymidine incorporation assay and cell counting. For the 3H-thymidine uptake, the cells were cultured in 24-well plates at 5 x 104 cells/well for 48 h in the presence of 17ßE2 (100 pM to 100 nM). During the last 18 h of incubation, cells were pulsed with 1 µCi 3H-thymidine. Then the cells were harvested, and samples were analyzed by ß-counter. The results were expressed as counts per minute per well (mean ± SE of three different experiments).
For cell counting, FNC-B4 cells were plated in 35-mm dishes (2 x 104 cells/dish) and allowed to adhere for 24 h. Different doses (100 pM to 100 nM) of 17ßE2 were added for 48 h, 72 h, or 6 d. The cells were also exposed to raloxifene or tamoxifen (100 pM to 100 nM) for 6 d. Then the cells were detached (trypsin 0.25%/EDTA) and cell number was determined by hemocytometer. The results were expressed as mean ± SE of three different experiments.
Viability assays: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Trypan blue dye exclusion
Cell viability after ß-amyloid exposure was determined by MTS assay (Promega Corp., Madison, WI), a colorimetric method for the determination of the number of viable cells in cytotoxicity assay. Cells were seeded in 96-well plates (1.5 x 104 cells/well) for 24 h. Thereafter 17ßE2, raloxifene, or tamoxifen (100 pM to 100 nM) was added for 3 or 6 d. During the last 18 h, 10 or 100 nM ß-amyloid was added to each well, and the assay was performed. Absorbance at 490 nm is directly proportional to the number of living cells in culture. The results were expressed in terms of mean ± SE viable cells/well in three different experiments.
Moreover, to assess the resistance against H2O2-mediated oxidative stress, viable cells were determined by Trypan blue dye exclusion test. Briefly, cells were cultured in 25-cm2 flasks with 17ßE2 (100 pM or 100 nM) for 6 d. During the last 20 h, 200 µM H2O2 was added. Subsequently cells were stained with Trypan blue dye for 1 min. Blue-positive and white-negative cells were counted in 10 fields (20x), and the results were expressed as mean ± SE of viable and dead cells/field in three different experiments.
hTERT expression
The absolute quantification of hTERT mRNA was performed by real-time RT-PCR, based on TaqMan technologies. The standard curve was generated with 5-fold serial dilutions (2.5 x 105 to 2.5 fg) of total RNA isolated from the human fibrosarcoma cell line HT1080 (American Type Culture Collection, Manassas, VA). Primers, probe, and RT-PCR conditions were as recently described by Yajima et al. (14). Because normalization to rRNA or glyceraldehyde-3-phosphate-dehydrogenase as well as other housekeeping genes has been clearly shown not to be accurate (15, 16), the results were expressed in terms of equivalent of femtograms HT1080 RNA per microgram total RNA. The experiments (n = 3) were run in triplicates.
Determination of apoptotic cells
Apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) analysis, using a commercially available detection kit (FragEL DNA fragmentation detection kit, Oncogene Research Products, Boston, MA). Briefly, in this assay terminal deoxynucleotidyl transferase binds to exposed 3'-OH ends of DNA fragments generated in response to apoptotic signals and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides. Biotinylated nucleotides are detected using a streptavidin-horseradish peroxidase conjugate, followed by diaminobenzidine. A dark brown signal indicates fragmented nuclei, which present pyknotic and roughly rounded or oval in shape. FNC-B4 cells were cultured in chamber slides in the presence or not of 100 pM 17ßE2 for 48 h. During the last 18 h, half of estrogen-treated and half of untreated chamber slides were exposed to 100 nM ß-amyloid. Four experiments were performed. Apoptotic cells per field were counted in 20 fields (50x), and the results were expressed as percentage of apoptotic cells per field (mean ± SE).
Additional experiments on apoptosis were performed by determining the amount of cleaved caspase-3 immunoreactive FNC-B4 cells. The cells were cultured in chamber slides in the presence or not of 100 pM 17ßE2 for 48 h in medium without phenol red. During the last 18 h, half of estrogen-treated and half of untreated chamber slides were exposed to 100 nM ß-amyloid. The cells were then washed with PBS and fixed in 3% paraformaldehyde for 20 min at room temperature. The immunocytochemical study was carried out using a polyclonal antibody against cleaved caspase-3 (Asp 175) (Cell Signaling Technology Inc., Beverly, MA) at 1:100 dilution in Tris-buffered saline/0.1% Triton X-100 for 24 h at 4 C. Subsequently incubation with a biotinylated secondary antibody (appropriately diluted in Tris-buffered saline/0.1% Triton X-100/5% BSA, 1:50) for 1 h was performed, and the reaction product was visualized by ABC peroxidase-based detection protocol and AEC kit (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA). Finally, the cells were counterstained with hematoxylin according to the manufacturer’s instructions. Apoptotic cells per field were counted in 10 fields (50x), and the results were expressed as percentage of apoptotic cells per field (mean ± SE).
Quantitative real-time RT-PCR for seladin-1 transcript
The measurement of seladin-1 transcript was also performed by quantitative real-time RT-PCR, based on TaqMan technologies. Total RNA was extracted from control cells as well as cells treated with different concentrations of 17ßE2, raloxifene, tamoxifen, PPT, or DPN for 72 h. Primers and probe were selected by the proprietary software Primer express (Applied Biosystems Inc., Foster City, CA). The sequences of seladin-1 primers, spanning 88 bp were: 5'-ATCGCAGCTTTGTGCGATG-3' (sense, exon 4, 5'-end at position 686); 5'-CACCAGGAAACCCAGCGT-3' (antisense, exon 5, 5'-end at position 774). The sequence of the probe, labeled with 6-carboxyfluorescein, which hybridized to the exon 4–5 junction region, was: 5'-TCCGTCCGAAAACTCAGACCTGTTCTATGC-3' (5'-end at position 708). The conditions of the real-time RT-PCR were the same as described previously (17). A calibration curve was generated using serial dilutions of a single-stranded sense oligodeoxynucleotide spanning the sequence included between the primers, as described by Bustin (15). The results were expressed as femtograms seladin-1 mRNA per microgram total RNA, for the reasons above discussed about hTERT expression. Three experiments were performed, and each experiment was run in triplicates.
Statistical analysis
Data were expressed as mean ± SE. Statistical differences were analyzed using one-way ANOVA. Significance was adjusted for multiple comparisons of means using Bonferroni’s approximation.
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Results |
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The stimulatory effect of 17ßE2 on DNA synthesis, as assessed by 3H-thymidine uptake, was statistically significant at the tested concentrations (100 pM to 100 nM) (Table 1
). With regard to cell duplication, a 48- or 72-h treatment with 17ßE2 (100 pM to 100 nM) did not affect cell number (Fig. 1). After a 6-d exposure to estrogen, a biphasic effect was observed. In fact, significantly increased cell counts were determined by exposure to 100 pM and 100 nM estrogen (151.9 ± 4.9 and 142.8 ± 5%, respectively, mean ± SE, vs. control, i.e. cells cultured in 1% FCS for 6 d, considered as 100%). Conversely, 1 and 10 nM 17ßE2 did not significantly affect cell number, compared with untreated cells, as shown in Fig. 1. Raloxifene and tamoxifen (100 pM to 100 nM) did not determine any effect on cell counts (data not shown).
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In the absence of estrogen/SERMs preincubation, ß-amyloid (10 and 100 nM) significantly and dose-dependently reduced cell viability (Fig. 2, A–C). Preincubation with 17ßE2, tamoxifen, or raloxifene (100 pM to 100 nM for 72 h) did not directly affect FNC-B4 cell viability. After estrogen exposure, neither 10 nM nor 100 nM ß-amyloid significantly altered cell viability (Fig. 2A). Noticeably, this effect was independent of cell proliferation rate, which was not increased by exposure to estrogen for 72 h, as mentioned above. The same results were obtained using tamoxifen (100 pM to 100 nM) (Fig. 2B) or raloxifene (100 pM and 1 nM) (Fig. 2C). Conversely, 10 and 100 nM raloxifene did not protect against ß-amyloid-induced toxicity (Fig. 2C). Virtually superimposable results were obtained in experiments in which the cells were preincubated with 17ßE2, tamoxifen, or raloxifene for 6 d (data not shown).
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Effect of 17ßE2 on ß-amyloid-induced apoptosis
Apoptotic rate in FNC-B4 cells was determined by TUNEL analysis. In control cells, the percentage of apoptotic nuclei was 7.25 ± 0.24%/field (mean ± SE) (Table 2). The exposure to 100 pM 17ßE2 did not change the amount of apoptotic cells (7 ± 0.25%/field, mean ± SE). Conversely, when FNC-B4 cells were exposed to 100 nM ß-amyloid, the percentage of cells undergoing apoptosis significantly increased (34 ± 0.48%/field, mean ± SE). However, when the cells were pretreated with estrogen (100 pM 17ßE2) before the addition of ß-amyloid, the effect of ß-amyloid as a proapoptotic factor was completely reverted (9 ± 0.25%/field, mean ± SE). In Fig. 4, A–D, the results of a typical experiment are shown. In Fig. 4A, control FNC-B4 cells are shown. No fragmented nuclei were detectable in the field. Conversely, apoptotic nuclei, represented by the dark dots and indicated by the arrows, were present in cells exposed to ß-amyloid (Fig. 4, B and C). Estrogen pretreatment effectively counteracted ß-amyloid-induced apoptosis, as shown in the field represented in Fig. 4D.
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). Estrogen treatment (100 pM 17ßE2) did not change the amount of positive cells (9.05 ± 3.01%/field, mean ± SE). The exposure to 100 nM ß-amyloid significantly increased the number of stained cells (40.57 ± 2.76%/field, mean ± SE). However, when the cells were pretreated with estrogen (100 pM 17ßE2) before the addition of ß-amyloid, caspase-3 activation was blunted (11.36 ± 2.04%/field, mean ± SE). In Fig. 5B, an example of the presence of immunostaining for cleaved caspase-3 after ß-amyloid exposure is shown. Conversely, no positive cells are present in the field shown in Fig. 5A (control cells) or Fig. 5C (17ßE2 pretreatment).
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The amount of seladin-1 mRNA was determined in untreated cells as well as in cells treated with different doses of 17ßE2 (from 10 pM to 100 nM for 72 h). The amount of seladin-1 mRNA in untreated cells was 112 ± 2.26 fg/µg total RNA (mean ± SE). 17ßE2 was able to significantly increase seladin-1 expression at all the concentrations that were used (Fig. 6). In addition, both raloxifene and tamoxifen (72-h exposure) significantly increased the level of seladin-1 expression (152.9 ± 5.1 and 176.4 ± 6.1%, mean ± SE, respectively, vs. control, considered as 100%, at 1 nM, as shown in Fig. 6), with the exception of raloxifene at high concentrations. In fact, remarkably, the exposure of FNC-B4 cells to 10 and 100 nM raloxifene, which did not exert neuroprotective effects (Fig. 2C
), markedly decreased the amount of seladin-1 mRNA (36.7 ± 0.5 and 44.1 ± 0.7%, mean ± SE, respectively), compared with untreated cells (data not shown). Finally, to determine whether estrogen-induced expression of seladin-1 was mediated by ER or ERß, FNC-B4 cells were treated with the ER selective agonist PPT or the ERß agonist DPN for 72 h at a concentration (10 nM) that has been previously reported to induce evident transcriptional activity (13). The results, also shown in Fig. 6, indicate that PPT determined a significant increase in the amount of seladin-1 mRNA (168.7 ± 4.9%, mean ± SE, vs. control), whereas DPN produced a weaker effect (128.2 ± 12%, mean ± SE, vs. control).
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Discussion |
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In this study, we addressed the neuroprotective role of estrogen and SERMs, using a unique human neuronal cell model (FNC-B4 cells). In fact these cells, isolated from human fetal olfactory epithelium, have been characterized as the precursors of olfactory receptor neurons, the only type of neuron in the olfactory epithelium (11). Moreover, FNC-B4 cells express both the ER and ERß (12). We found that estrogen treatment significantly increased cell duplication rate, yet selectively at 100 pM and 100 nM and only after a prolonged exposure (6 d). Conversely, the SERMs raloxifene and tamoxifen had no effect on the cell number at any of the concentrations tested (100 pM to 100 nM). In addition, exposure of FNC-B4 cells to 17ßE2 (100 pM to 100 nM) dramatically increased cell resistance to ß-amyloid toxicity, completely counteracting the markedly reduced cell viability caused by ß-amyloid in untreated cells. It is worth noting that the neuroprotective effect of estrogen was obtained starting from a 72-h treatment, which did not have any effect on cell proliferation. Therefore, estrogen-mediated neuroprotection appears to be independent of the rate of cell duplication.
Interestingly, a similar neuroprotective effect was obtained with exposure to the same concentrations of tamoxifen. Raloxifene was also able to protect against the toxic effect of ß-amyloid but only at low concentrations (100 pM and 1 nM). This finding is in agreement with the results of another very recent study (21), in which, depending on the concentration of the drug, mixed agonist-antagonist effects of raloxifene in rat neurons were observed. Moreover, our data showed that estrogen readily counteracted the effect of oxidative stress. The expression of the hTERT gene, the catalytic subunit of telomerase, which contains a functional estrogen-responsive element in its promoter region (22), was also assessed and found to be markedly increased by 17ßE2 exposure. This finding is in agreement with experimental evidence showing that brain telomerase activity, which is virtually present only in neuroblasts and early postmitotic embryonic neurons (reviewed in Ref.23), mediates cell survival-promoting actions of brain-specific factors, such as the brain-derived neurotrophic factor (24). Noteworthy, the possibility to protect neurons against age-related degeneration by manipulating telomerase activity in the neural stem cells present in the adult brain has been considered as a new possible frontier of the exploding field of stem cell research (reviewed in Ref.25).
In our study, we also evaluated the effect of estrogen on apoptosis in FNC-B4 cells. TUNEL analysis as well as the detection of the amount of cleaved caspase-3 clearly demonstrated that 17ßE2 treatment completely reverted the proapoptotic effect of ß-amyloid. Therefore, according to previous data, yet using a peculiar human neuronal cell model, our results seem to support the hypothesis that estrogen exerts a neuroprotective action by conferring resistance to toxic factors and suggest that SERMs may also have a similar role. This is indeed a novel and very interesting finding because, whereas SERMs actions in breast, bone, and uterus have been well characterized, their effects in the brain are considerably less well understood. Although an ideal SERM has yet to be developed, in theory it should have antagonistic activity in the breast and uterus and agonistic effect in the bone, cardiovascular system, and brain. Our results are in agreement with recent studies highlighting the concept that, in addition to estrogen, SERMs have neuroprotective effects. In fact, for instance, tamoxifen and raloxifene were able to protect the brain in ischemia-reperfusion models of ischemic stroke (reviewed in Ref.26). In addition to neuroprotective effects, there is increasing evidence that SERMs may also be neurotrophic. Tamoxifen increased synaptic density in the hippocampus of ovariectomized rats, and raloxifene stimulated neurite outgrowth in pheochromocytoma PC12 cells (reviewed in Ref.26).
In view of the recently reported neuroprotective effects of seladin-1 (6), we hypothesized that it might be a new downstream effector of estrogen in neurons. Using a quantitative technique optimized in our laboratory (17), we found detectable levels of seladin-1 transcript in FNC-B4 cells. Noticeably, the amount of seladin-1 mRNA was significantly increased by estrogen treatment at all the concentrations tested, paralleling its neuroprotective effects. This is the first report demonstrating that the expression of seladin-1 gene is up-regulated by estrogen. Moreover, both raloxifene and tamoxifen also determined a similar increase of the amount of seladin-1 mRNA, in agreement with the observed neuroprotective effects. Nevertheless, a peculiar finding in our experimental model was that the stimulatory effect of raloxifene on seladin-1 expression was limited to low concentrations of the molecule. In fact, high concentrations (10 and 100 nM) did not increase but markedly decreased the amount of seladin-1 mRNA. Because of the parallel lack of a neuroprotective effect of raloxifene at the same concentrations, this finding strongly suggests that seladin-1 is directly involved in mediating the neuroprotective effects of estrogen/SERMs in FNC.
Overall, our original results, obtained using an original in vitro model, are in agreement with previous data, which appear to support a role for estrogen therapy in AD. With regard to this point, despite the lack of a clear demonstration that estrogen effectively arrests the progression of the disease, hormonal therapy appeared to have beneficial effects on learning and memory and was associated with a reduced risk for AD in several studies (reviewed in Ref.5). It has been hypothesized that the response to treatment may depend on several factors, such as age, menopausal status, and preexisting risk factors (i.e. smoking, apolipoprotein E genotype) (27). In particular, there seems to be a critical time for estrogen treatment. In fact, early and prolonged therapy has been found to produce the maximum benefit in terms of reduced risk for AD (28, 29).
A debated question is whether the protection conferred by classical nuclear ERs is mediated by the - or the ß-subtype or both. Although the results from ER knockout (ERKO) and ERß knockout (ßERKO) mice are somewhat controversial, it is worth noting that, whereas 17ßE2 exerted a protective effect in the brain of ovariectomized ßERKO mice, it did not in ERKO mice. This strongly suggests a critical role for ER in neuroprotection (30). Furthermore, it is worth mentioning that decreased expression of ER in hippocampal neurons of AD patients has been observed (31). However, a possible role for ERß in neuroprotection has been postulated, based on the evidence that ßERKO mice undergo increased neuronal loss throughout life, compared with wild-type controls (32). Because FNC-B4 cells express both the ER and ERß, we performed additional work to demonstrate whether the induction of seladin-1 expression by estrogen occurred via the ER or ERß. We found that in our cell model a selective ER agonist (PPT) determined a much stronger increase of seladin-1 mRNA than an ERß agonist (DPN), thus indicating that the expression of this neuroprotective factor is mainly controlled by ER stimulation. With regard to estrogen targets, it should also be mentioned that recent findings suggest that the brain contains a plethora of ERs, differently expressed in relationship to specific sites and developmental stages. In addition to nuclear ER and ERß, recently a third member of the ER family, ER
, has been identified (33). Furthermore, a variety of nuclear, cytoplasmic, and plasma membrane ERs has been described in the brain, including for instance G protein-coupled receptors, a novel membrane-associated ER-X and a truncated ER variant (reviewed in Ref.34). The finding that SERMs, similarly to estrogen, may also act through nongenomic pathways (reviewed in Ref.26), further suggests that these molecules play an important role in brain physiology and highlights the need for additional experimental studies covering this topic.
In summary, in the present study, using an original human cell model, we add evidence that estrogen/SERMs may confer protection to neuronal cells, and we suggest for the first time that this effect may be mediated, at least in part, by increased expression of the recently identified gene seladin-1. The observation that in our experimental model the neuroprotective effect elicited by hormone exposure may be independent of proliferation rate and is strictly related to seladin-1 expression appears to be in full agreement with this hypothesis. Nevertheless, additional studies, designed to silence seladin-1 expression, should further confirm the involvement of this gene in the response of neurons to estrogen/SERMs.
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Acknowledgments |
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Footnotes |
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First Published Online December 7, 2004
Abbreviations: AD, Alzheimer’s disease; DPN, diarylpropionitrile; 17ßE2, 17ß-estradiol; ER, estrogen receptor; ERKO, ER knockout; ßERKO, ERß knockout; FCS, fetal calf serum; FNC, fetal neuroepithelial cells; hTERT, human telomerase reverse transcriptase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PPT, propylpyrazole-triol; seladin-1, selective AD indicator-1; SERM, selective estrogen receptor modulator; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
Received January 14, 2004.
Accepted November 23, 2004.
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References |
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24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet 69:685–694[CrossRef][Medline]
- and ß-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Mol Cell Endocrinol 206:13–22[CrossRef][Medline]
, not ß, is a critical link in estradiol-mediated protection against brain injury. Proc Natl Acad Sci USA 98:1952–1957 expression in hippocampal neurons in relation to hyperphosphorylated tau in Alzheimer patients. Acta Neuropathol 106:213–220[CrossRef][Medline]
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