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and ß Turnover in Cultured Human Uterine Artery Endothelial Cells
Department of Obstetrics and Gynecology, Division of Gynecological Endocrinology and Reproductive Medicine, University of Vienna Medical School, General Hospital, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Walter Tschugguel, M.D., University of Vienna Medical School, Department of Obstetrics and Gynecology, Division of Gynecological Endocrinology and Reproductive Medicine, General Hospital, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: walter.tschugguel{at}akh-wien.ac.at.
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Abstract |
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expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER and/or ERß levels is unclear. Using RT-PCR assays, we did not find any regulation of ER or ERß mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17ß-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17ß-estradiol was capable of down-regulating both ER and ERß protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER, but not ERß, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER as well as ERß through different proteasomal uptake mechanisms. |
Introduction |
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Estrogens influence gene expression, growth, and cellular differentiation in target cells by activating one or both of two estrogen receptors (ER), ER and ERß (4, 5). ERs have been studied intensely in female reproductive physiology, but functional ERs are also present and physiologically important in other tissues of both sexes, including the brain, bone, liver, skin, and the cardiovascular system (6).
Regulation of ER concentration is a key component in limiting estrogen responsiveness in target cells. The level of ER in cells changes with varying physiological states. In most cases, the primary endocrine regulator is the ligand itself. In an autoregulatory feedback loop, estrogen induces a decline in both ER protein and mRNA (7). Recently, a nongenomic action of estrogen that involves nuclear ER has been described, whereby rapid proteolysis of ER protein occurs via a proteasome-mediated pathway (8, 9). The ubiquitin-proteasome system is responsible for the degradation of many of the short-lived proteins in eukaryotic cells. The pathway targets proteins for degradation by the proteasome via covalent tagging of the substrate protein with a polyubiquitin chain. The multi-ubiquitinated substrate protein is then degraded by the 26 S proteasome in an ATP-dependent reaction (10). In contrast to ER, however, there are no data available on whether or not ERß could also be a target of ligand-mediated turnover.
ER and ERß are both expressed in vascular endothelial and smooth muscle cells (11). Vascular ERß mRNA expression was found to be increased after balloon injury in mice aorta (12), and abnormal vascular function as well as hypertension was shown in mice deficient in ERß (13). Yet the mechanisms governing ER and ERß concentration in vascular endothelial cells are undetermined.
In the present study, we used cultured human uterine artery endothelial cells (HUAEC) as well as a MCF-7 breast cancer cell line to explore regulation of one or both of the two ERs, ER and ERß, by estrogen.
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Materials and Methods |
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Vessel segments of the uterine artery were excised within 3 h after hysterectomy and immersed in ice-cold (4 C) Hanks’ balanced salt solution (Sigma, St. Louis, MO) containing antibiotics (penicillin, streptomycin, and fungizone, BioWhittaker, Inc., Verviers, Belgium) and 5% fetal calf serum (FCS, Pan Biotech, Aidenbach, Germany). Surrounding tissue was removed, and vessel segments were cut open and incubated in 0.2% collagenase type IV (Pan Biotech) at 37 C for 10–20 min to detach endothelial cells from the surface. Cell suspension was filtered through a cell strainer with 100-µm pores (Becton Dickinson and Co., Franklin Lakes, NJ). Cells were washed with Hanks’ balanced salt solution containing 5% FCS (wash solution) at 4 C, centrifuged for 5 min at 1500 x g at 4 C, and resuspended in wash solution. The cell suspension was then mixed with M 450 rat-antimouse-mouse-antihuman CD 31 dynabeads for 10 min and washed five times with cold wash solution to remove unbound cells. Cells with beads were resuspended in M199 medium containing 20% FCS with antibiotics, heparin (5 U/ml; Immuno, Vienna, Austria), endothelial cell growth supplement (40 µg/ml; Technoclone Inc., Vienna, Austria), and L-glutamine (2 mmol/liter; BioWhittaker, Inc.). Cells, denominated HUAEC (14), were grown and passaged at 37 C under 5% CO2/95% air. HUAEC were subcultured using a split ratio of 1:2. The endothelial origin was confirmed by typical cobblestone morphology, immunofluorescence staining with antibodies against von Willebrand factor, angiotensin converting enzyme, and Ulex Europaeus I lectin, as well as uptake of acetylated low-density lipoprotein. The medium was replaced every 2 d.
Confluent passage four cells were maintained in phenol red-free medium containing 10% charcoal-filtered FCS for at least 48 h before induction with hormones. Cells were then treated with 17ß-estradiol (E2) for various times and concentrations. Additionally, 30 min before E2, cells were treated with the partial ER antagonist tamoxifen (TAM, 10-6 M), insulin (10-6 M), the proteasome inhibitor lactacystin (25 µM), and the protein synthesis as well as proteolysis inhibitors cycloheximide (CHX, 50 µM) and puromycin (PUR, 50 µM), or respective vehicles for agonists.
Western analysis
Nuclear and cytoplasmic fractions were prepared from cells using a commercially available nuclear and cytoplasmic extraction reagent kit (NE-PER, Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. Protein concentrations were measured by spectrophotometry using the MicroBCA-Protein Assay Reagent from Pierce Chemical Co.
A total of 25 µg of nuclear extracts were used for experiments, and 1 µg of a recombinant human (rh) ER protein (Affinity BioReagents, Inc., Golden, CO) or a rhERß protein (Alexis Biochemicals, San Diego, CA) was used as positive control for detection of ER and ERß protein, respectively. After quantification, proteins were electrophoresed by the use of SDS-PAGE on ExcelGel 8–18% gradient gels (Amersham Pharmacia Biotech, Uppsala, Sweden) and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked in PBS containing 2.5% nonfat dry milk, 2.5% BSA (Sigma), and 0.05% Tween 20 (Promega Corp., Madison, WI) for at least 1 h. Immunoreactions were performed with either an anti-ER (AB-10, Neomarkers, Fremont, CA) or an anti-ERß (6B12, Genetex, San Antonio, TX) monoclonal antibody (1 µg/ml each). A monoclonal mouse IgG1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1 µg/ml instead of the primary antibodies as negative control for each antibody. Antibody to I
B (C21, Santa Cruz Biotechnology, Inc.) was used to visualize protein not regulated by estrogen in endothelial cells and as a loading control (15). The antibody hybridization was followed by a horseradish peroxidase-conjugated goat antimouse-IgG (40 ng/ml; Pierce Chemical Co.). Specific reaction products were detected by the SuperSignal West Pico Chemoluminescent Substrate kit (Pierce Chemical Co.) according to the manufacturer’s instructions.
Assay of in vitro ER breakdown in MCF-7 and HUAEC nuclear extracts
To investigate whether turnover of ER and/or ERß in nuclear extracts from untreated HUAEC or MCF-7 cells (positive control) was through the ATP-ubiquitin proteolytic pathway, we performed in vitro ER and ERß protein degradation assays using a previously described method (16) that was slightly modified as follows: the reaction mixture contained, in a final volume of 50 µl, 10 µg nuclear extract of MCF-7 cells and 30 µg nuclear extract of HUAEC, 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 3 mM dithiothreitol, 4 mM ATP, 10 mM creatine phosphate, 5 µg creatine phosphokinase (ATP-generating system), and either 4 µg ubiquitin (Sigma) or 40 µg methyl-ubiquitin (Sigma) for 2 h at 37 C. Reactions were separated by boiling samples in the presence of SDS-loading buffer [100 mM Tris-HCl (pH 8.0)/200 mM dithiothreitol/4% SDS/20% glycerol/0.2% bromophenol blue]. The reaction mixtures were resolved by 8–18% SDS-PAGE, and reaction products were visualized by Western analysis using the ER antibodies described above.
RT-PCR
RNA extraction and reverse transcription. Pure total RNA was extracted from cultured endothelial cells by isopyknic centrifugation and treated with DNAse I (Roche Molecular Biochemicals, Indianapolis, IN) as described (17). cDNA was synthesized in 50 µl total volume containing the commercially available Random Primed Reverse Transcription Reaction Mix (ViennaLab, Vienna, Austria), 40 U RNasin (Promega Corp.), 400 U µ-MLV Reverse Transcriptase (ViennaLab), and 1 µg total RNA. Reactions were incubated at room temperature for 10 min, followed by 50 min at 37 C and 5 min at 95 C.
DNA amplification.
PCRs were performed on a Perkin-Elmer GeneAmp PCR System 2400 (Perkin-Elmer Corp., Norwalk, CT). PCR was performed in a total volume of 25 µl containing 3 µl cDNA template, 25 pmol of each primer (all primer sequences and mapping positions are listed in Table 1
; primers for ER are located in exons 4 and 6; primers for ERß are located in exon 4), 250 µM deoxynucleoside triphosphates, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.01% (wt/vol) gelatin, 1.5 mM MgCl2, 0.1% Triton X-100, and 0.5 U Super Taq DNA Polymerase (ViennaLab). To control for errors in input of cDNA used in PCRs, amplification of the ubiquitous ß-2-microglobulin cDNA was performed in parallel using ß-2-microglobulin-specific primers. The amplification profiles were as follows: 94 C for 30 sec, 50 C (ß-2-microglobulin and ERß) or 48 C (ER) and 72 C for 30 sec; 35 (ER and ERß) or 22 (ß-2-microglobulin) cycles. To ensure that these PCR systems work in a quantitative way, all of them were thoroughly optimized with respect to their exponential amplification phase. This was done by using different reaction times, reaction temperatures, and cycle numbers as well as different concentrations of reaction components. To simplify the performance and to increase the reproducibility of PCR, PCR master mixes containing primers, deoxynucleoside triphosphates, and buffer were prepared and used in all amplification reactions. In addition, tubes containing all PCR components and distilled water instead of cDNA served as negative controls to check for the presence of DNA that may have been carried over from prior reactions. All PCRs were performed at least twice in separate experiments.
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, 213 bp; ERß, 130 bp; ß-2-microglobulin, 231bp) were separated on agarose gels [5–10 µl PCR product; 3% SB Fine Gel Agarose (Severn Biotech Ltd., Kidderminster, UK)] and visualized by performing the electrophoresis on SYBR Green I (Molecular Probes, Inc., Eugene, OR) -containing gels.
Quantitative real-time RT-PCR.
cDNA was synthesized from total RNA as described above. The amplification primers and the TaqMan probe (Table 2) for the ERß real-time PCR were designed with the Primer-Express software (PE Applied Biosystems, Foster City, CA). The ERß forward primer binds in exon 1, and the ERß reverse primer binds in exon 2. FAM (6-carboxyfluorescein) was used as the reporter dye, and TAMRA (6-carboxy-tetramethylrhodamine) was used as the quencher dye. Oligonucleotide synthesis and purification were done by VBC-Genomics Bioscience Research GmbH (Vienna, Austria). The reaction was performed in 25 µl total volume containing 4 µl cDNA, 25 pmol of each amplification primer, 5 pmol probe, and 12.5 µl 2x TaqMan Universal Mix (PE Applied Biosystems). The reaction conditions were 50 C for 2 min, 95 C for 10 min (activation of the AmpliTaq-Gold Polymerase), and then 40 cycles of 15 sec at 95 C (denaturation) followed by 60 sec at 60 C (annealing and extension). To correct variations linked to differences in the amount of RNA taken for the reaction or to different levels of inhibition during RT or PCR, we normalized the ERß expression using the ß-2-microglobulin gene, a ubiquitously expressed housekeeping gene, as a reference gene. The expression of this housekeeping gene was quantified with the ß-2-microglobulin Control Reagents Kit from PE Applied Biosystems, according to the manufacturer’s guidelines. All ERß and ß-2-microglobulin experiments were performed in triplicate, and several negative controls were included. Fluorescence emission was continuously monitored and analyzed by a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems) with the GeneAmp 5700 SDS Software (version 1.1).
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For the generation of ERß and ß-2-microglobulin standard curves, we used 2-fold serial dilutions of the HUAEC cDNA sample, which showed the highest ERß mRNA expression levels in ERß real-time RT-PCR, as templates. Standard curves were constructed and calculated by the GeneAmp 5700 SDS software referring the threshold cycle (PCR cycle at which a specific fluorescence becomes detectable) to the log of the cDNA starting quantity of each dilution step. These standard curves allowed us to interpolate the unknown ERß and ß-2-microglobulin mRNA expression levels in each analyzed sample.
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Results |
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A rhER and a rhERß protein as well as extracts from separated nuclear fractions of HUAEC were subjected to standard SDS electrophoresis, transferred to a membrane, and probed with ER and ERß antibodies. The results shown in Fig. 1 demonstrate the presence of an immunoreactive protein fraction with an apparent molecular mass of approximately 67 kDa recognized by the ER antibody with the rhER protein (Fig. 1A), the HUAEC or the MCF-7 extract, but not the rhERß protein (Fig. 1A), whereas an immunoreactive protein with a molecular mass of approximately 55 kDa was recognized by the ERß antibody with the rhERß protein as well as with the HUAEC and MCF-7 extract, but not with the rhER protein (Fig. 1B). However, a somewhat larger protein band was detectable within both HUAEC and MCF-7 extracts compared with rhERß protein. These apparent differences may reflect protein species microheterogeneity in posttranslational modifications, such as phosphorylation (18). Parallel experiments with isotype monoclonal antibodies failed to produce a distinctive band in these regions and confirmed the specificity of the 67- or 55-kDa proteins toward both ER antibodies used (data not shown).
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The cytosolic fractions from HUAEC equally lacked or exhibited only a faint immunostained band (data not shown). E2 at a concentration such as that attained during the ovulatory phase of the menstrual cycle (10-10 M) down-regulated both ER and ERß protein after 1 h (Figs. 2–5) and 2 h (data not shown) of E2 administration, whereas a 30-min pretreatment with TAM blocked both ER and ERß degradation (Figs. 2–5). The amount of down-regulation was comparable at either concentration of E2 used in these experiments (Figs. 2 and 3).
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Rapid proteolysis of ER, at least in MCF-7 cells (9) and lactotrope cells of the anterior pituitary (8) has recently been shown to occur mainly via a proteasome-mediated pathway. We therefore assessed the potential implication of the proteasome complex in E2-mediated ER and ERß elimination in HUAEC. Remarkably, lactacystin (a highly specific, irreversible proteasome inhibitor; Ref.19) abrogated both ER and ERß elimination (Figs. 4
and 5). By contrast, insulin, a naturally occurring inhibitor of cellular proteolysis, acting at least in part by decreasing the ubiquitin-mediated proteasomal activity of its target cells (20), abrogated E2-induced ER, but not ERß, elimination in HUAEC (Figs. 4 and 5), suggesting that ER but not ERß turnover is a target of insulin action in human vascular endothelial cells.
Addition of 50 µM CHX to the medium 30 min before E2 treatment abrogated the effect of E2 on ER but not ß (Figs. 4 and 5), establishing the involvement of protein synthesis in vascular endothelial cell ER, but not ERß, turnover. Of note, when CHX was replaced by PUR, which also blocks ER down-regulation (21), a similar decrease of ER turnover was observed (data not shown), refuting the possibility of potential artifact associated with the use of only one protein synthesis/proteolysis inhibitor. To further verify that the decrease in protein was specific for ER, blots were reprobed with antibody against the ubiquitous protein, IB, which is not regulated by estrogen. IB protein levels were unchanged in the presence and absence of E2 and, thus, served as both a loading control for total protein content and a standardization of ER levels (Fig. 4).
From that data, a major difference in the metabolic degradative pathways following binding of E2 to ER or ERß should be considered.
ATP-dependent degradation of ER in MCF-7 and HUAEC nuclear extracts
In an effort to examine the role of the ubiquitin-proteasome pathway in ER and/or ERß degradation, we used a cell-free system modified from Hershko et al. (16). With that system, we aimed at discovering whether or not endogenous MCF-7 ER degradation, which has been reported to occur via proteasome-mediated proteolysis (8, 9), MCF-7 ERß degradation, and HUAEC ER and/or ERß degradation are ubiquitin dependent. For that purpose, we examined the specificity of the ATP-dependent degradation in nuclear extracts by using wild-type ubiquitin, or an inhibitor directed at the ubiquitin-substrate conjugation reactions, methylated ubiquitin (MeUb). Proteins are tagged for recognition by the 26 S proteasome via the conjugation of a polyubiquitin chain to the targeted protein (10). This polyubiquitin chain is generated by isopeptide linkages between the carboxy terminus of each ubiquitin molecule with lysine 48 of the preceding ubiquitin. This process is competitively inhibited by MeUb (22). Thus, MeUb was added in an amount recently shown to be sufficient for inhibition of MyoD degradation in HeLa nucleoplasm (23) to overcome the effect of endogenous ubiquitin. As seen in Fig. 6, the ATP-dependent degradation of both ER and ERß is inhibited by MeUb in both MCF-7 cells and HUAEC to a different extent. This difference might be most attributable to the lesser amount of intrinsic nuclear degradation machinery within endothelial nuclear extracts compared with MCF-7 nuclear extracts, however this remains to be clarified. This inhibition is not seen by the addition of wild-type ubiquitin in either case. These data indicate that the ubiquitin-proteasome pathway is responsible for the ATP-dependent degradation of both ER and ERß in MCF-7 breast cancer cells, as well as in HUAEC.
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To examine a potential involvement of E2-mediated ER turnover at the level of transcription, total RNA was extracted from cells treated with indicated E2 concentrations for 1 h (Fig. 7) or 2 h (data not shown) or with TAM 30 min before 10-10 M E2, and cell extracts were then analyzed by means of RT-PCR for ER mRNA (Fig. 7) and ERß mRNA (data not shown) as well as quantitative real-time RT-PCR for ERß mRNA expression (data not shown). We did not find any difference in the amount of ER and ERß mRNA expression levels after E2 administration in HUAEC.
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Discussion |
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The aim of this study was to determine the influence of the natural estrogen, E2, on the concentration of ER and ERß in human vascular endothelial cells. Our main result is that even low concentrations of E2 induce a substantial down-regulation of vascular endothelial ER as well as ERß protein starting after 1 h of incubation with E2. This down-regulation on the protein level is not accompanied by a similar decrease in the amount of respective mRNA, indicating that transcription is not involved in that process. These data suggest that ER is implicated in targeted down-regulation in vascular endothelial cells that is comparable to targeted ER down-regulation in HeLa cells (25), MCF-7 breast cancer cells (9), as well as lactotrope cells of the anterior pituitary (8), by the natural ligand estrogen. Moreover, our data introduce ERß as a target of ligand-mediated down-regulation, at least in endothelial cells. We next aimed to explore whether the partial estrogen receptor antagonist TAM is able to affect ER and ERß turnover in endothelial cells. We found TAM acting as an antagonist of E2-mediated ER and ERß degradation in our cells. In addition to its effect to relax precontracted rabbit coronary arteries (26), our finding that TAM increases ER and ERß bioavailibility in vascular endothelium may also contribute to the evidence that TAM is associated with a reduction in the incidence of fatal myocardial infarction in women (27).
According to previous data exhibiting the proteasome pathway as the major system for selective ATP-dependent nongenomic degradation of ER by the natural ligand estrogen (8, 9, 25), we asked whether inhibitors of the proteasome pathway were able to reduce the in vitro degradation of ER and/or ERß in vascular endothelium. First, we found lactacystin, an irreversible proteasome inhibitor as markedly effective to inhibit ER as well as ERß degradation, demonstrating that both vascular endothelial ER and ERß protein are turned over through the proteasome pathway. Second, we found insulin, a naturally occurring, reversible inhibitor of protein degradation (28), as effective to inhibit ER, but not ERß, turnover in endothelial cells. Insulin promotes cellular growth and maintenance by a wide variety of anabolic and anticatabolic actions, including the inhibition of overall proteolysis (28). However, the proteolytic systems regulated by insulin are unclear. We now demonstrate that ER but not ERß, turnover is a target of insulin inhibition in our endothelial cells. Although not shown here, this selective effect of insulin might most likely be due to activation of a signaling pathway that inhibits ubiquitin ligase activity, which is specific for ER, but not ERß. However, experiments are needed to reject or validate this point of view.
In addition to being insensitive to insulin, we found that ERß turnover is not compromised after treatment with CHX and PUR that have recently been implicated to compromise ER turnover in MCF-7 cells through proteolysis inhibition, but not protein synthesis inhibition (21). This led us to hypothesize the presence of at least two proteasomal uptake mechanisms existing in our endothelial cells, which differ according to the ER implicated: a mechanism blocked by inhibition of proteolysis, which emerges in the presence of CHX, PUR, and insulin, specific for ER bioavailibility; and a mechanism unaffected by proteolysis inhibition, specific for ERß turnover. Previous work suggested that CHX may block the synthesis of an enzyme involved in the cascade of events leading to ubiquitination of ER (9). We therefore attempted to explore whether ubiquitin contributes to ER and/or ERß turnover in vascular endothelium. For that purpose, we used a cell-free system consisting of nuclear extracts from either MCF-7 cells, used as a control system for turnover of ER, or uterine artery endothelial cells. Interestingly, we found the degradation of ER as well as that of ERß in MCF-7 cells and HUAEC dependent on ubiquitin. The findings on ER turnover correspond with previous data obtained from within MCF-7 cells (8, 9). In addition to ER, we now introduce ERß as a target of the ubiquitin-proteasome system, at least in MCF-7 breast cancer cells and HUAEC. However, the mechanisms by which inhibition of proteolysis selectively operates on ER, but not ERß, deserves further clarification.
Based on our findings, according to the nature of ER involved, we propose the existence of two different ubiquitin-dependent proteasomal uptake mechanisms that compromise estrogen receptor turnover in human vascular endothelial cells. It is likely that both systems contribute to selective modulation of ER dynamics in uterine artery vascular endothelium, dependent on the orchestrated nature of a wide variety of vasoactive agonists.
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Footnotes |
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Abbreviations: CHX, Cycloheximide; E2, 17ß-estradiol; ER, estrogen receptor; FCS, fetal calf serum; HUAEC, human uterine artery endothelial cell; MeUb, methylated ubiquitin; PUR, puromycin; TAM, tamoxifen.
Received July 29, 2002.
Accepted February 6, 2003.
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B-dependent mechanism. J Cell Biochem 73:321–331[CrossRef][Medline]
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