Multiple forms of estrogen receptor-alpha in cerebral blood vessels: regulation by estrogen

Chris Stirone, Sue P. Duckles, and Diana N. Krause

Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697-4625


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The cerebral vasculature is an important target tissue for estrogen, as evidenced by significant effects of estrogen on vascular reactivity and protein levels of endothelial nitric oxide synthase and prostacyclin synthase. However, the presence, localization, and regulation of estrogen receptors in the cerebral vasculature have not been investigated. In this study, we identified the presence of estrogen receptor-alpha (ER-alpha ) in female rat cerebral blood vessels and localized this receptor to both smooth muscle and endothelial cells by use of immunohistochemistry and confocal microscopy. With immunoblot analysis, multiple forms of ER-alpha were detected at 110, 93, 82, 50, and 45 kDa in addition to a relatively weak band corresponding to the 66-kDa putative unmodified receptor. The 82-kDa band was identified as Ser118-phosphorylated ER-alpha , whereas the 50-kDa band lacks the normal NH2 terminus, suggestive of an ER-alpha splice variant. Lower molecular mass bands persisted after in vivo inhibition of 26S proteasome activity with lactacystin, whereas the 110- and 93-kDa bands increased. All forms of ER-alpha in cerebral vessels were decreased after ovariectomy but significantly increased after chronic estrogen exposure in vivo.

estrogen receptors; 26S proteasome; 17beta -estradiol; cerebral circulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A GROWING BODY OF EVIDENCE suggests that estrogen may produce its protective effects in the cerebral vasculature, in part by modulating the synthesis and release of endothelial-derived vasodilators. Previous work in our laboratory demonstrated that, in cerebral blood vessels, chronic estrogen exposure leads to elevated protein levels of endothelial nitric oxide synthase (eNOS), cyclooxygenase-1, and prostacyclin synthase, enzymes that produce the vasodilators nitric oxide (NO) and prostacyclin (26, 32). In contractile studies that used rat and mouse cerebral arteries, effects of chronic estrogen treatment depended entirely on an intact endothelium, suggesting that estrogen modulates cerebrovascular reactivity through an action on the endothelium, with no effect on smooth muscle reactivity per se (9, 10). The effect of estrogen treatment on levels of vasodilator-producing enzymes in cerebral arteries is thought to be receptor mediated, because estrogen treatment of estrogen receptor-alpha (ER-alpha ) knockout mice does not elevate these proteins (11). In addition, preincubation of female rat cerebral blood vessels with the ER antagonist ICI-182780 or partial agonist tamoxifen completely prevents estrogen-dependent increases in eNOS protein in vitro (27).

Actions of estrogen in target tissues are mediated by two defined receptor subtypes, ER-alpha and ER-beta . These receptors and their respective signal transduction pathways and regulatory mechanisms are therefore a subject of intense research. ER-alpha , first cloned in 1986, was believed to mediate all of the physiological effects of estrogen until 1995, when ER-beta was cloned from rat prostate (12, 13, 23, 31). Both subtypes of ER have been detected in vascular tissue (41). Various estrogen target tissues have been probed for the presence of ER-alpha by immunoblot analysis, and most tissues display the putative 66-kDa receptor. However, additional bands of ER-alpha immunoreactivity have been detected at molecular masses ranging from 28 to 112 kDa (3, 4, 15, 25, 28, 36, 37). These bands, distinct from 66 kDa, have been attributed to unique states or splice variants of ER-alpha or to proteolysis products, either from proteases or proteasomal degradation. However, the functional significance of these multiple bands in any estrogen target tissue is not yet well understood.

The cerebral circulation is regulated by a variety of mechanisms, including myogenic responses, pH and PCO2, metabolic factors, and vascular innervation (6). It is also clear that estrogen treatment has a profound effect on the function of this vascular bed (34). However, little is known concerning the presence, localization, and regulation of ERs in the cerebral vasculature. Because there is evidence that ER-alpha is important for estrogen modulation of cerebral vascular reactivity, we used confocal microscopy to characterize the cell-type localization of ER-alpha . We also used immunoblot analysis to characterize ER-alpha proteins in cerebral vessels and assess possible regulation of these receptors by estrogen treatment.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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In vivo treatments. All animal procedures were approved by the Institutional Animal Care and Use Committee. Fischer 344 female rats (3 mo old, Harlan Sprague Dawley) were unoperated (INT.F), ovariectomized (OVX), or ovariectomized and treated with 17beta -estradiol (OE), as described previously (32). Rats were anesthetized by intraperitoneal injection of 46 mg/kg ketamine and 4.6 mg/kg xylazine for all surgical procedures. OE animals received a subcutaneous silicone implant (placed dorsally at the neck) filled with 17beta -estradiol at the time of ovariectomy. Implants were left in place for 4 wk; animals were then anesthetized by CO2 and killed by decapitation. Brains and ovaries were isolated and used immediately or frozen at -80°C. We have previously demonstrated by this procedure that serum estrogen levels in OE animals are within the physiological range (32). Body weights were 184 ± 1 g for OVX and 164 ± 1 g for OE. Uterine weights were 33 ± 2 mg for OVX and 127 ± 4 mg for OE (P <=  0.05). In some cases, OVX and OE rats received a single intraperitoneal injection of 1 mg/kg lactacystin (Affinity Bioreagents) in 0.9% saline vehicle (17). Animals were euthanized at 2, 6, or 12 h after injection, as indicated, and cerebral vessels were isolated as described in the following section. Cerebrovascular proteasome activity was measured using a kit from BIOMOL.

Cerebral vessel isolation. Pial arteries were dissected from the surface of the brain. Alternatively, blood vessels were isolated from whole brain as described previously (26, 32). Briefly, four brains from each treatment group were pooled, gently homogenized in a Dounce tissue grinder in ice-cold phosphate-buffered saline (PBS), and then centrifuged at 720 g for 5 min at 4°C. The supernatant was discarded, and the pellet was resuspended and washed in cold PBS twice, followed by 720 g centrifugation for 5 min at 4°C. The resuspended pellet was then layered over 16% dextran (35-45 kDa, Sigma) followed by centrifugation at 4,500 g for 20 min at 4°C to pellet the blood vessels. The supernatant was discarded, and the parenchymal tissue layer was resuspended in PBS and layered again over dextran, followed by centrifugation at 4,500 g for 20 min at 4°C. The pellets were combined in cold PBS, and blood vessels were harvested and washed over a 50-µm nylon mesh. This preparation contains both pial and intraparenchymal vessels that, examined with light microscopy, are a mixture of arteries, arterioles, capillaries, veins, and venules.

Confocal microscopy. Cerebral blood vessels were dissected from the surface of the brain, cut into small segments, fixed in 3% formaldehyde for 30 min, and permeabilized using Triton X-100 (0.1%) for 5 min. Vessels were then incubated for 30 min in 1% bovine serum albumin (BSA)-PBS and then incubated overnight at 4°C in primary antibody at 1:50 dilution [rabbit anti-ER-alpha H-184 (Santa Cruz) and mouse anti-eNOS-610296 (Transduction Laboratories)]. Vessels were then washed for 30 min in PBS (with change in buffer every 10 min) and incubated overnight at 4°C with the secondary antibodies at 10 µg/ml (goat anti-rabbit Oregon green 488 and goat anti-mouse Texas red, Molecular Probes), followed by a final wash for 30 min (buffer change every 10 min) in PBS. Vessels were then laid on slides and covered with mounting medium (VectaShield, Vector Laboratories), and coverslips were applied. Images were obtained using a Bio-Rad model 1024 laser scanning confocal microscope equipped with standard and UV lasers.

Immunoblot analysis. Blood vessels were glass homogenized at 4°C in lysis buffer (50 mM beta -glycerophosphate, 100 µM NaVO3, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1 mM DL-dithiothreitol, 20 µM pepstatin, 20 µM leupeptin, 0.1 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. Samples were then centrifuged for 10 min at 4,500 g at 4°C, and the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Lysates were used immediately or stored at -80°C. In all immunoblot experiments, equal amounts of protein [50 µg in 1× SDS sample buffer (Invitrogen), boiled for 4 min] were loaded in each lane of an 8% Tris-glycine gel (Novex) and separated by SDS-PAGE. Positive controls consisted of recombinant human ER-alpha (Panvera) and rat ovary lysate. Molecular masses of immunoreactive bands were determined by loading biotinylated molecular mass standards (Bio-Rad). After electrophoresis, protein was transferred to nitrocellulose membranes (Amersham) and subsequently incubated at 4°C overnight in blocking buffer (0.01 M PBS, 0.1% Tween 20, and 6.5% nonfat dry milk). Membranes were then incubated with primary antibodies (HC-20, H-184, p-Ser118, Santa Cruz; PA1-308, Affinity BioReagents) at dilutions from 1:500 to 1:200 for 3 h at room temperature, followed by 5× 5-min washes in 0.1% Tween 20-PBS (T-PBS). Blots were then incubated in secondary antibody [goat-anti-rabbit IgG-horseradish peroxidase (HRP) for H-184, PA1-308, and HC-20; donkey-anti-goat IgG-HRP for p-Ser118, Santa Cruz] at 1:10,000 dilution for 1 h at room temperature. Biotinylated standards were incubated in 1:7,500 streptavidin-HRP (Sigma). Both segments of the membrane were then washed 5× 5 min in T-PBS. Membranes were then incubated with enhanced chemiluminescence reagent (Amersham) for 1 min and exposed to Hyperfilm (Amersham) for 15 s to 10 min, depending on signal strength. Software for electrophoresis analysis, UN-SCAN-IT (Silk Scientific), was used for densitometric analysis of immunoreactive bands.

Statistics. Values for body and uterine weights are means ± SE. Statistical differences in immunoblot band densities were determined by one-way ANOVA followed by Dunnett's Multiple Comparison Test (GraphPad Prism 2.0 software) and displayed as a fold difference relative to OVX or vehicle. In all cases, statistical significance was set at P <=  0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Localization of ER-alpha in cerebral blood vessels. To observe the presence and localization of ER-alpha in cerebral vessels, a rabbit polyclonal antibody generated against the first 185 amino acids of the NH2 terminus of ER-alpha (H-184) was used to probe small segments of pial vessels isolated from female rat brain. The nuclear stain 4',6-diamidino-2-phenylindole dihydrochloride was used to identify smooth muscle cell nuclei (concentric to the vessel circumference) or endothelial cell nuclei (parallel to direction of blood flow) (2). With use of an anti-rabbit secondary antibody conjugated to Oregon green 488 and an anti-mouse secondary antibody conjugated to Texas red, laser scanning confocal microscopy revealed intense immunoreactivity for ER-alpha (green) in smooth muscle cells (Fig. 1B). No reactivity for eNOS (red), a marker for endothelial cells, was detected in smooth muscle (Fig. 1, A and C). When the vessels were visualized at the level of the endothelium near the internal elastic lamina, strong immunoreactivity for eNOS (red) was detected (Fig. 1D). Strong ER-alpha immunofluorescence (green) was detected in the same focal plane (Fig. 1E). Figure 1F shows the merged image of Fig. 1, D and E, revealing strong colocalization of ER-alpha with eNOS in the endothelial layer (orange).


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Fig. 1.   Laser scanning confocal microscopy was used to image a small segment of a pial artery isolated from female rat brain. The artery was dual-stained with an NH2-terminal estrogen receptor (ER)-alpha antibody (H-184, green) and an anti-endothelial nitric oxide synthase (eNOS) antibody (red). A and B represent a single focal plane through the smooth muscle layer, imaged for eNOS and ER-alpha , respectively. C represents a merging of A and B. D and E show the same vessel segment, but in a different focal plane through the endothelial cell layer, imaged for eNOS and ER-alpha , respectively. F represents a merging of D and E.

Western blot analysis with various antibodies to ER-alpha . Two different cerebral vessel preparations were compared by using immunoblot analysis for ER-alpha with both COOH-terminal (HC-20) and NH2-terminal (H-184) antibodies. Pial vessels from freshly isolated brains were quickly dissected and immediately homogenized at 4°C in lysis buffer and compared with lysate from vessels isolated from whole brain with the density centrifugation procedure. When run in adjacent lanes for comparison, no differences in the location or number of ER-alpha immunoreactive bands were observed between surface vessels isolated by rapid dissection and whole brain vessels isolated by density centrifugation with either antibody (Fig. 2, COOH-terminal antibody shown). With either vascular preparation, little to no immunoreactivity was observed at 66 kDa, which corresponds to the unmodified ER-alpha . Detection of immunoreactivity at 66 kDa was found to require loading >= 50 µg of total protein, an antibody dilution of 1:100-1:200, and lengthy film exposure; therefore, this band was not consistently seen. However, strong immunoreactivity was consistently observed in both preparations at ~110, 93, 82, and 50 kDa under normal immunoblot conditions. Protease inhibitors were present in the lysis buffer in both cases, and immediate boiling/freezing of protein lysates in SDS sample buffer did not alter the immunoreactive bands detected. Multiple freeze-thaw cycles of recombinant human (r) ER-alpha also did not show any change in bands detected (data not shown). Furthermore, as an additional control, rER-alpha was added to cerebrovascular protein lysate and run under SDS-PAGE conditions adjacent to an equal amount of rER-alpha alone and to protein lysate without rER-alpha . Addition of rER-alpha produced a strong band at 66 kDa, consistent with the rER-alpha control, but other bands in the lysate were unchanged (data not shown). Therefore, we concluded that the major band detected at ~50 kDa was not due to cleavage of the native receptor by proteases or to freeze-thawing of protein lysates.


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Fig. 2.   Representative Western blot using a COOH-terminal antibody to ER-alpha (HC-20) to compare freshly dissected pial vessels (A) and whole brain vessels isolated under density centrifugation (B), both from intact female rats. Blot shown is representative of blots probed with either NH2-terminal (H-184, n = 3) or COOH-terminal (HC-20, n = 3) antibodies to ER-alpha .

Because there was no difference in ER-alpha immunoreactive bands seen in immunoblots from rapidly dissected pial arteries and vessels isolated from whole brain by density centrifugation, the latter isolation procedure was used for further studies. Cerebral vessel lysates from OVX, OE, and intact female rats were compared for the presence of ER-alpha protein by use of a rabbit polyclonal antibody generated against the last 20 amino acids of the COOH terminus of ER-alpha (HC-20) (Fig. 3A). Rat ovary lysates were also run for comparison. In all cases, weak immunoreactivity was detected at 66 kDa, which corresponds to the positive control (rER-alpha ). Vessels from all animal groups showed the additional bands at ~110, 93, 82, and 50 kDa. All of these immunoreactive bands showed greater density than the 66-kDa band. Similar bands were detected in lysate from ovary, an estrogen target tissue known to express ER-alpha (5).


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Fig. 3.   Cerebral blood vessels from ovariectomized (OVX), 17beta -estradiol-treated OVX (OE), and intact female (INT.F) rats were probed for ER-alpha immunoreactivity and compared with two positive controls, recombinant human ER-alpha (rER-alpha ) and rat ovary lysate. A: COOH-terminal antibody to ER-alpha (HC-20). Western blot is representative of 6 separate experiments. B: NH2-terminal antibody to ER-alpha (H-184). Blot is representative of 6 separate experiments. C: antibody recognizing phosphorylated Ser118 ER-alpha . rER-alpha was not detected by this antibody and is not shown. Blot is representative of 4 separate experiments.

To determine whether any of these bands are due to nonspecific binding of the COOH-terminal antibody, a blocking peptide for HC-20 was preincubated with the primary antibody before immunoblot analysis. All of the previously described immunoreactive bands disappeared after pretreatment with the blocking peptide (data not shown).

To further validate these immunoblot results, a different antibody generated against amino acids 1-185 of the NH2 terminus of ER-alpha (H-184) was used. As shown in Fig. 3B, with this antibody immunoreactivity was detected at 66 kDa, but again only at relatively low levels. More reactive individual bands were detected at ~110, 93, 82, and 50 kDa.

Protein bands running higher than 66 kDa, seen with antibodies to either the COOH or NH2 terminus of ER-alpha , suggested that ER-alpha might exist in vivo with posttranslational modifications such as ubiquitination or phosphorylation. A recent study showed that ER-alpha phosphorylated at Ser118 had reduced electrophoretic mobility under SDS-PAGE conditions (18). Therefore, we used an antibody specific for the Ser118-phosphorylated ER-alpha to test whether any of the bands identified by COOH- or NH2-terminal antibodies might contain phosphorylated Ser118 ER-alpha . As shown in Fig. 3C, with use of an antibody specific for Ser118-phosphorylated ER-alpha , a single immunoreactive band was detected at an apparent molecular mass of 82 kDa. This result suggests that the 82-kDa band seen with both the NH2- and COOH-terminal antibodies can be attributed to Ser118-phosphorylated ERalpha .

In cerebral vessels, unmodified 66-kDa ER-alpha was present in only small quantities compared with all other ER-alpha immunoreactive bands. The major band detected ran at ~50 kDa, close to the predicted molecular mass of the A/B domain-deleted isoform of ER-alpha , 46 kDa (35). Interestingly, a band at ~45 kDa could also be discerned in some blots produced with antibodies H-184 and HC-20 as part of a doublet of ~50 and 45 kDa (see Figs. 5A and 7). Because the A/B domain-deleted isoform of ER-alpha lacks the first ~170 amino acids of the NH2 terminus, we tested this hypothesis by using an antibody generated against the first 21 amino acids of the NH2 terminus of ER-alpha (PA1-308, Affinity Bioreagents). As shown in Fig. 4, little to no reactivity to this antibody was seen at ~50 kDa in either the ovary or cerebral vessels isolated from whole brain of intact female rats. This indicates that the NH2 terminus is missing in the ~50-kDa band, supporting the idea of an A/B domain-deleted ER-alpha variant. The relatively strong bands seen previously at 93 and 82 kDa were also lacking in blots with this antibody. In the ovary, the PA1-308 antibody detected a strong band at 66 kDa. Little reactivity was seen at 66 kDa in cerebral vessels. Moderate reactivity was seen, however, in both vessels and ovary at 110 and 45 kDa.


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Fig. 4.   An NH2-terminal antibody recognizing only the first 21 amino acids of ER-alpha (PA1-308) was used to probe lysate of cerebral blood vessels isolated from intact female rat brains. Positive controls were rER-alpha and ovary lysate. Western blot shown is representative of 3 separate experiments.

Inhibition of the 26S proteasome. Several in vitro studies have shown that ER-alpha is ubiquitinated and subsequently degraded by the 26S proteasome (1, 15, 21, 30). To validate this degradation pathway for cerebral vessels and also to test the hypothesis that the strongly immunoreactive band at 50 kDa resulted from proteasomal degradation, intact female rats received a single intraperitoneal injection of the 26S proteasome inhibitor lactacystin (1 mg/kg) and were euthanized 2, 6, or 12 h later. To verify that in vivo lactacystin treatment inhibited cerebrovascular proteasome activity, an assay for proteasome activity was performed. Cerebrovascular proteasome activity was inhibited by 94% 2 h after intraperitoneal injection of lactacystin [vehicle = 21,524 ± 212 arbitrary fluorescence units (AFU); 2-h treated = 1,197 ± 39 AFU; n = 3]. As shown in Fig. 5, immunoreactivity of both the 93- and 110-kDa bands significantly increased after lactacystin treatment. This effect was seen within 2 h of lactacystin injection and was maintained, with minimal further increase, at 6 and 12 h. The 110-kDa band displayed a robust sixfold increase, whereas an approximately twofold increase was seen in the immunoreactive band at 93 kDa. Interestingly, the density of the 82-kDa band showed only a minor increase, which was not significantly different from vehicle-treated levels. No increase in immunoreactivity could be detected for the putative receptor at 66 kDa; any band at this molecular mass was below the limit of detection in all blots from lactacystin-treated vessels. However, a very faint band was detected at 66 kDa in vessels from the vehicle-treated animals. In contrast, the density of the 50-kDa band was not reduced but was maintained at 6 and 12 h after proteasomal inhibition, indicating that it is not a degradation product of the 66-kDa ER-alpha .


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Fig. 5.   Effect of proteasome inhibition with lactacystin on ER-alpha immunoreactive bands. Intact female rats were given ip injections of vehicle (V) and euthanized 2 h later, or were given lactacystin (1 mg/kg) and euthanized 2, 6, or 12 h later. Whole brain vessels were isolated using density centrifugation and probed for ER-alpha using either NH2- or COOH-terminal antibodies. A: representative Western blot with the COOH-terminal antibody (HC-20) shows immunoreactive bands from cerebral vessel lysates. Lane 1, recombinant ER-alpha . Blot shown is representative of 3 separate experiments. B: densitometric analysis of major bands detected using either COOH- or NH2-terminal antibody (HC-20, H-184) shows change in ER-alpha protein levels after lactacystin treatment as fold increase compared with vehicle. Because immunoreactive patterns were identical with each antibody, results were pooled for this analysis. Values are means ± SE. * Significantly different from vehicle (P <=  0.05).

Effect of 17beta -estradiol exposure on ER-alpha . As shown in Fig. 6, lysates from animals chronically exposed to estrogen (OE and intact females) contained significantly greater amounts of ER-alpha immunoreactive protein compared with OVX. As shown in Fig. 3, A and B, this difference was seen using antibodies against either the COOH or NH2 termini in all immunoreactive bands and was not limited to an effect of estrogen on the putative receptor at 66 kDa. The two identified bands, 66 and 82 kDa, were quantified. Figure 6A shows that, in cerebral vessels from both intact females and OE animals, there is an ~1.5-fold greater level of protein at 66 kDa, as detected with the NH2-terminal antibody, compared with vessels from OVX animals. When the antibody specific for the Ser118-phosphorylated state of ER-alpha was used, there also was an ~1.5-fold greater level of immunoreactivity in vessel protein from animals exposed to estrogen compared with OVX (Fig. 6B).


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Fig. 6.   Effect of estrogen status on density of selected ER-alpha immunoreactive bands. Cerebrovascular protein lysates from OE, OVX, and intact female rats (INT.F) were compared. Results are expressed as fold increase in OE and INT.F vs. OVX. Values are means ± SE. * Significantly different from OVX (P <=  0.05). In each case, a representative blot is shown as well as mean data from densitometric analysis (n = 3). A: 66-kDa band with an NH2-terminal antibody (H-184). B: 82-kDa band with an antibody to Ser118-phosphorylated ER-alpha .

It is currently not clear whether ligand binding stabilizes ER-alpha or, alternatively, promotes ER-alpha degradation (40). Our data in vessels from OE animals are consistent with the idea of ER-alpha stabilization by the presence of estrogen. Therefore, to determine the role of estrogen, if any, in modulating proteasomal degradation of ER-alpha , vessels from OVX and OE animals were compared 2 h after administration of the proteasome inhibitor, when immunoreactivity of higher molecular weight bands was maximally elevated. As seen in Fig. 7, lactacystin was effective in elevating ER-alpha immunoreactivity in cerebral blood vessels from both OVX and OE animals.


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Fig. 7.   Effect of estrogen status on response to lactacystin administration. OVX and OE animals were given ip injections of either vehicle (V) or lactacystin (1 mg/kg), and ER-alpha protein levels were compared after 2 h of exposure. Each 2-h cerebral artery lysate represents a separately treated animal. A representative Western blot using a COOH-terminal antibody (HC-20) is shown. Illustrated experiment was repeated twice (n = 2 for vehicle; for lactacystin treatment: n = 4 for OE, n = 4 for OVX).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown that ER-alpha is present in cerebral blood vessels and is localized to both smooth muscle and the endothelium. Surprisingly, very little unmodified, 66-kDa receptor could be detected in female rat cerebral vessels by use of immunoblot analysis. However, major immunoreactive bands for ER-alpha were detected at ~45, 50, 82, 93, and 110 kDa. Immunoreactivity at 82 kDa corresponded to the Ser118-phosphorylated state of ER-alpha . The ~50-kDa band is not a degradation product but does lack the normal ER-alpha NH2 terminus, suggestive of an ER-alpha splice variant (35). Furthermore, we have shown that estrogen exposure elevates protein levels of all ER-alpha immunoreactive bands. In addition, we demonstrate the importance of the 26S proteasome in mediating degradation of ER-alpha in cerebral vessels.

The presence of ER-alpha in smooth muscle and endothelial cells of cerebral vessels was expected, as estrogen has been shown to have direct effects on the vasculature (27). These effects include increased protein levels of the enzymes eNOS and prostacyclin synthase (26, 32), which produce the vasodilatory molecules NO and prostacyclin, respectively. Although not demonstrated in the cerebral vasculature, estrogen is also known to act on vascular smooth muscle cells to inhibit proliferation (29). The aforementioned effects of estrogen on vascular tissue are likely the result of classic nuclear hormone receptor-induced transcriptional activation of genes, often referred to as "genomic" effects. However, there is a growing body of evidence suggesting that estrogen may also exert "nongenomic" effects on vascular tissue, defined as effects that occur too rapidly to be explained by the genomic mechanism (7, 14, 22, 37-39). These nongenomic effects have been seen primarily in endothelial cells in culture and include rapid production and release of NO and activation of the mitogen-activated protein (MAP) kinase pathway. It is interesting to note that, in all cases cited, the ER antagonist ICI-182780 and partial agonist tamoxifen blocked these rapid effects, further validating that the effects of estrogen on vascular tissue are mediated by one of the two previously characterized estrogen receptors, alpha  or beta , or a receptor(s) highly homologous to ER-alpha and -beta .

Multiple bands of ER-alpha immunoreactivity. The predicted molecular mass of the cloned unmodified ER-alpha is 66 kDa (31). Very little unmodified ER-alpha was detected in cerebral blood vessels. Instead, multiple bands of both higher (82, 93, and 110 kDa) and lower (45 and 50 kDa) molecular masses were observed in pial and whole brain vessel preparations. Use of multiple antibodies and specific blocking peptides further indicate that the multiple immunoreactive bands were due to specific ER-alpha antibody interactions. Unfortunately, many published blots of ER-alpha do not show the entire molecular mass range; therefore, it is impossible to know whether these authors have observed, or ignored, some of the bands we have reported. However, several studies in estrogen target tissues, such as mouse uterus, rabbit uterine microsomes, breast tumor cells, and mouse cerebral cortex, have reported ER-alpha immunoreactive bands at a wide range of molecular masses, including 45-50, 66, 92-94, and 110-112 kDa (3, 15, 25, 28, 36). Many of these bands appear at molecular masses identical to those we found in cerebral blood vessels. Therefore, additional experiments were performed to attempt to characterize some of the ER-alpha immunoreactive bands found in cerebral vessels.

Serine118-phosphorylated ER-alpha . One band consistently detected by immunoblot analysis in cerebral vessels runs at an apparent molecular mass of 82 kDa. Our data indicate that this band corresponds to the Ser118-phosphorylated state of ER-alpha . The electrophorectic shift of Ser118-phosphorylated ER-alpha appears greater in our blots than that described previously (18). A possible explanation is that ER-alpha may be polyubiquitinated after phosphorylation, a mechanism that has been described for progesterone receptors (24). Although increased phosphorylation of ER-alpha in response to estradiol is well documented (31), phosphorylation of Ser118 and subsequent enhancement of transcription by the activiated AF-1 region were originally reported as being estrogen independent (31). The literature currently reflects that both estrogen and activators of the MAP kinase pathway can lead to phosphorylation of ER-alpha at Ser118 (31). The mechanism by which estrogen increases Ser118-phosphorylated ER-alpha in cerebral vessels is unknown. However, one possible explanation is that the increase seen in total ER-alpha protein levels in the presence of estrogen simply increases the amount of receptor available for phosphorylation and is not necessarily due to induction of phosphorylation by estrogen of the MAP kinase pathway. A similar conclusion has been drawn with respect to phosphorylation of the androgen receptor (20).

Immunoreactivity at ~50 kDa. One of the most prominent ER-alpha immunoreactive bands in cerebral arteries appeared at ~50 kDa. Our positive control, rat ovary lysate, showed all of the same ER-alpha immunoreactive bands as in cerebral vessels, including a dense band at 50 kDa. Other workers, using a COOH-terminal antibody for ER-alpha and human umbilical vein endothelial cells, also showed strong immunoreactivity at 45 kDa and weak reactivity at 66 kDa, a ratio similar to that seen in our study (37). In many studies, ER-alpha immunoreactive bands lower than 66 kDa have been suggested to result from proteolysis. In our study, we took care to prevent proteolysis by routine use of protease inhibitors, and we also demonstrated that our method of tissue isolation did not contribute to the appearance of additional ER-alpha immunoreactive bands. Our original hypothesis to explain this ~50-kDa band was that unmodified (66-kDa) ER-alpha was being cleaved in the NH2-terminal region by the proteasome, removing the first ~140-170 amino acids. Our data indicate, however, that neither the strong immunoreactive band at ~50 kDa nor the ~45 kDa band is a product of proteasomal degradation, as they remained unchanged after 12 h of in vivo proteasome inhibition. Additionally, no concomitant increase in the 66-kDa band was detected at any measured time point after lactacystin exposure.

Recent work in rat pituitary demonstrated the presence of naturally occurring isoforms of ER-alpha generated through differential splicing of the ER-alpha primary transcript (33). Two of these splice variants have predicted molecular masses of 53 and 45 kDa. In COS-7 cells transfected with unmodified ER-alpha , a 45-kDa band also was shown to result from an alternative translation site in the DNA sequence and not receptor proteolysis (4). A 46-kDa isoform of human ER-alpha that is created by alternative splicing of the primary gene was recently identified in MCF-7 cells, and it is missing the first ~170 amino acids from the NH2 terminus (8). Interestingly, in cerebral vessels, the antibody targeted to the first 20 amino acids of the NH2 terminus of ER-alpha (PA1-308) did not detect the ~50 kDa band, suggesting that this band may be a truncated form of ER-alpha lacking the NH2-terminal A/B domain. Work done in EA.hy926 cells suggests that a 45-kDa splice variant may be responsible for rapid, nongenomic responses (NO release) to 17beta -estradiol stimulation (37). Thus the prominent 50-kDa band in cerebral vessels may be a variant form of ER-alpha with possible unique signaling mechanisms.

Proteasomal degradation. Our findings suggest that there is a steady-state degradation of ER-alpha in cerebral vessels. These results indicate that half-lives of some forms of ER-alpha in cerebral vessels may be on the order of ~2-6 h, as maximal or near-maximal increases in both 110- and 93-kDa ER-alpha immunoreactive bands were seen after 2 or 6 h of proteasome inhibition with lactacystin. However, longer time points may be required to see increases in both the 82- and 50-kDa bands, because in our study protein levels of these two bands from lactacystin-treated vessels were not significantly different from levels in vehicle-treated controls.

Effect of estrogen on ER-alpha . A surprising finding in this study is that chronic estrogen exposure increased ER-alpha protein levels. All forms of ER-alpha were decreased by ovariectomy and increased by estrogen treatment. Although desensitization and downregulation are common mechanisms involving many ligand-activated receptors, regulation of nuclear hormone receptors is not so well understood. For example, androgen receptor (AR) half-life is stabilized over sixfold in the presence of ligand in AR-transfected COS cells (20). In female mice, ovariectomy reduced ER-alpha levels in cortex (3). However, both progesterone receptors and ER-alpha have been reported to show a decreased half-life or downregulation in the presence of ligand (1, 21, 24). Interestingly, a recent study suggested that, in the absence of estrogen, ER-alpha is highly ubiquitinated, offering another explanation for the differences seen in ER-alpha levels in OVX vs. OE animals in our study (40). Analysis of Fig. 7 might suggest that estrogen has no effect on levels of ER-alpha in the absence of proteasomal degradation. However, this experiment was designed to determine only maximal effects of lactacystin treatment; therefore, further experiments are needed to verify this point. In fact, if estrogen binding stabilizes ER-alpha , protecting it from degradation, this could account for the increased ER-alpha immunoreactivity observed in vessels from estrogen-treated animals. Most work of this nature has been done in cell culture, where gene regulation and signal transduction may not reflect in vivo conditions. Our findings are important, because they reflect in vivo effects of chronic estrogen exposure on the regulation and state of ER-alpha in cerebral blood vessels.

In summary, we have demonstrated that ER-alpha is present in cerebral blood vessels and is localized to both smooth muscle and endothelium. Multiple antibodies generated against both the NH2 and COOH termini of ER-alpha detect identical immunoreactive bands of molecular masses both higher and lower than the putative 66-kDa ER-alpha . These bands appear to be unique states or splice variants of ER-alpha , as freeze-thaw cycling of vessel lysates, addition of protease inhibitors, or in vivo proteasome inhibition did not alter immunoreactivity below 66 kDa. Immunoreactivity at 50 kDa is eliminated when an antibody against the first 20 amino acids of the NH2 terminus is used, indicating that the full A/B domain is likely deleted, supporting the idea of a previously described splice variant of ER-alpha (4, 8). Furthermore, increases seen in higher molecular mass ER-alpha immunoreactivity after proteasome inhibition indicate that these proteins are regulated by proteasomal degradation, consistent with what has been described for ER-alpha in the literature (1, 21, 30).

The increases seen in ER-alpha protein levels due to chronic estrogen exposure may offer a secondary mechanism by which estrogen may produce protective effects in the cerebral vasculature. Elevated ER-alpha protein levels may provide a reserve of receptors by which estrogen can mediate increases in enzyme levels responsible for the increased production of endothelial-derived vasodilators, overcoming the steady-state degradation of ER-alpha mediated by the 26S proteasome. Finally, a better understanding of the unique states and isoforms of ER-alpha that appear to be present in cerebral vessels and their relevant genomic and nongenomic signal transduction mechanisms may lead to novel preventive and therapeutic approaches to the treatment of stroke and cardiovascular disease.


    ACKNOWLEDGEMENTS

We thank Jonnie Stevens for technical assistance with surgical procedures and Anabel Martinez and Amin Boroujerdi for help with cerebral vessel isolation.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant RO1-HL-50775 and by a grant from the American Heart Association.

Address for reprint requests and other correspondence: D. N. Krause, Dept. of Pharmacology, College of Medicine, Code 4625, Univ. of California, Irvine, CA 92697-4625 (E-mail: dnkrause{at}uci.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 11, 2002;10.1152/ajpendo.00165.2002

Received 18 April 2002; accepted in final form 4 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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