Published online before print June 24, 2004, doi: 10.1161/01.RES.0000136521.70093.f1
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Differential and Opposing Regulation of PAI-1 Promoter Activity by Estrogen Receptor 
and Estrogen Receptor ß in Endothelial Cells
From the Departments of Medicine and Pharmacology, Vanderbilt University Medical Center and Nashville Veterans Affairs Medical Center, Nashville, Tenn.
Correspondence to Douglas E. Vaughan, MD, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 383 PRB, 2220 Pierce Ave South, Nashville, TN 37232-6300. E-mail doug.vaughan{at}mcmail.vanderbilt.edu
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Abstract |
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To investigate the molecular mechanisms involved in the estrogen-dependent control of plasminogen activator inhibitor-1 (PAI-1) gene expression in vascular cells, we compared the transactivation properties of estrogen receptors (ER
and ERß) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ER increased PAI-1 promoter activity in BAECs by an estrogen-dependent mechanism, whereas ERß suppressed PAI-1 promoter activity by an estrogen-independent mechanism. The suppressive activity of ERß was dominant over the inductive activity of ER. Mutation of a putative estrogen response element (ERE) located at position –427 in the proximal promoter abolished the ER action without influencing the suppressive effects of ERß. Mutation of either AP1-like site did not eliminate the ER or ERß actions at the PAI-1 promoter, suggesting that other promoter elements are involved in these responses. These mutations significantly reduced the –3.4kbp PAI-1 promoter response to serum. We concluded that ER and ERß exert differential effects on the PAI-1 promoter activity in transfected BAECs. ER activated the PAI-1 promoter through a proximal ERE (–427) and possibly additional EREs located within the PAI-1 promoter, whereas ERß suppressed the promoter construct via an unidentified mechanism. This is the first demonstration of the differential regulation of a vascular gene promoter by ER and ERß.
Key Words: estrogen receptors • plasminogen activator inhibitor-1 promoter • estrogen response element • bovine aortic endothelial cell
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Introduction |
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Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor family and contributes to the regulation of endogenous fibrinolysis by irreversibly binding both tissue-type and urokinase plasminogen activators. Substantial experimental and epidemiological evidence illustrates that PAI-1 may in fact contribute to the development of ischemic cardiovascular disease.1,2 The well-documented ability of estrogen to reduce plasma levels of PAI-13,4 provides a compelling reason to further explore the molecular mechanisms through which estrogen regulates PAI-1.
The effects of estrogen on the human vasculature are complicated and controversial. While the Nurses Health Study suggested that hormone replacement therapy significantly reduced the relative risk of myocardial infarction or cardiovascular death,5,6 both the Heart and Estrogen/Progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI), however, suggested that hormone replacement therapy increased thrombotic complications.7–10 On a molecular level, the cardiovascular effects of estrogen are thought to reflect the complex actions of estrogen on lipid metabolism, nitric oxide synthesis, fibrinolytic balance, and extracellular matrix production.11–16 The mechanisms by which estrogen mediates these various effects are not clearly understood but likely involve direct effects on gene expression in the vasculature.
The concept of a direct vascular protective effect of estrogen is supported by several experimental findings17–19 and may be mediated via endothelial estrogen receptors (ERs).20,21 Two forms of the ER, ER, and ERß, function as ligand-dependent transcription factors.22,23 Although ER and ERß have many structural and functional similarities, subtle differences in these receptor activities may account for the variable actions of estrogen in various tissues.24 In cells that express both receptors, ERß appears to function as a dominant-negative regulator of ER at subsaturating concentrations of ligand but has no effect on ER-mediated transcription at saturating concentrations of ligand.25–27 ERs can influence gene transcription in at least 2 ways. ERs can directly bind estrogen response elements (EREs) in the promoters of estrogen-regulated genes,28 or they can form heterotrimeric transcriptional complexes with transcription factors, such as AP-1,29 Sp1,30 or CREB to regulate gene expression indirectly.31 ER and ERß have been shown to induce synthetic ERE-driven reporter constructs in response to multiple ligands.32 The principle physiological ligand, 17ß-estradiol, activates AP-1–dependent transcription by ER but inhibits AP-1–dependent transcription via ERß,33 suggesting that 17ß-estradiol regulates ER-dependent and ERß-dependent expression similarly via ERE, but differently via AP-1 response elements.29
In this study, we explored the abilities of candidate cis-activating and trans-activating components of the estrogen response system that control PAI-1 promoter activity in a bovine aortic endothelial cell (BAEC) model system. The data presented here demonstrate the differential effects of ER and ERß in transactivating the human PAI-1 promoter. We also show that ER action at the PAI-1 promoter is partially dependent on a newly identified ERE located between nucleotides –427 and –407 in the PAI-1 promoter.
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Materials and Methods |
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Cell Culture
BAECs were isolated and maintained as described previously.34 For transfections, cells (passaged 4 to 8 times) were subcultured to 60% to 70% confluence in phenol red-free media supplemented with 10% delipidated fetal bovine serum in 12x25-mm well plates coated with 2% gelatin.
Plasmid Constructs
The plasmid constructs –3.4kbp PAI-1 and –0.8kbp PAI-1 are genomic fragments of human PAI-1 promoter region cloned into the pGL2 basic vector (Promega, Madison, Wis) as described previously.35 The plasmid, pER, was generated by ligating a human ER cDNA EcoRI fragment into pcDNA3.1. The plasmid, pERß, is a murine ERß cDNA fragment ligated into pcDNA3.1 (Invitrogen, Carlsbad, Calif). The mouse ERß shares 88.5% nucleotide identity with the human ERß, but shares 93% similarity at the amino acid level, as determined using EMBOSS–Align needle method.36
Polymerase Chain Reaction–Based Site-Directed Mutagenesis
For polymerase chain reaction–based site-directed mutagenesis of the P-box (proximal AP1-like site) and the D-box (distal AP1-like site), in the –3.4kbp PAI-1 promoter construct, the following primer sets were generated, with bolded nucleotides representing the mutated region: P-box mutant primer, sense strand, (5'-CGGACTCCCAGAGCCAGCCTAGGGGTGGGGCTGGAACATGAG-3'); P-box mutant primer, antisense strand, (5'-CTCATGTTCCAGCCCCACCCCTAG CTGGCTCTG GGAGTCCG-3'); D-box mutant primer, sense strand, (5'-GTGGGTGGGGCTGGAACA CCT AGG CATCTATTTCCTGCCCACATC-3'); and D-box mutant primer, antisense strand, (5'-GA TGTGGGCAGGAAATAGATGCCTAGGTGTTCCAGCCCCACCCAC-3').
For site-directed mutagenesis of the ERE37 in the –0.8kbp PAI-1 construct and in the –3.4kbp PAI-1 promoter construct, the following primer set was generated, with bolded nucleotides representing the mutated ERE half-site: ERE mutant primer, sense strand, (5'-TATCTTTGATAA CTCCACAAAGCTTTGGTTCGCCAAAGGAAAAGC-3'); and ERE mutant primer, antisense strand, (5'-CCTTTTCGTTTGGCGAACCAAAGCTTTGTGGAGTTATC AAAGATA-3'). Polymerase chain reaction-based site-directed mutagenesis was performed as described.38 The sequence integrity of the mutated promoter regions was confirmed by automated sequencing and restriction endonuclease analysis.
Transfection Analyses
Transfections were performed in phenol red-free low-serum media using lipofectamine-plus as directed by the manufacturer’s protocol (Invitrogen, Carlsbad, Calif). For each independent experiment, transfections for each well were performed in triplicate with the following amounts of plasmid DNAs: –3.4kbp PAI-1 (0.4 µg) or –0.8kbp PAI-1 (0.4 µg) plus pcDNAHisLacZ (0.2 µg) plus pcDNA3.1 (0.1 µg), pER (0.1 µg), or 0.1 µg pERß (0.1 µg), unless otherwise indicated. Transfections were allowed to proceed for 3 hours at 37°C followed by replacement of the lipofectamine–DNA complex with media containing 10% delipidated fetal bovine serum and supplemented with either vehicle (0.1% wt/vol ethanol) or estrogen (100 nmol/L). The transfected cells were incubated an additional 48 hours before assaying for luciferase activity or beta-galactosidase activity as described previously. Raw luminometer units were normalized to reference luciferase activity. PAI-1 promoter activities are reported as mean relative induction levels with ±standard error of the mean (SEM).
Electromobility Shift Assay
The reactions were performed as described previously 39,40 with hER and mERß, in vitro translated proteins (TNTquick Transcription Translation System; Promega, Madison, Wis). Protein samples were pre-incubated for 5 minutes on ice with 1.0 µg of poly(dI/dC) (Pharmacia Biotech, Upsala, Sweden) and 1 µg of single-stranded nonspecific oligonucleotide in a reaction buffer containing 10 mmol/L Tris pH 7.4, 100 mmol/L KCl, 10% glycerol, 1 mmol/L EDTA, 1 mmol/L DTT, 0.3 µg bovine serum albumin, 0.1% Triton-X 100, and 5 mmol/L MgCl2 before the addition of the radiolabeled probe. The unlabeled competitor oligonucleotide was added during preincubation. An oligonucleotide (5'-CCAGGTCAGAG TGACCTGAGCTAAAAT-3') that contains the PAI-1 putative ERE motif was used to assay the binding capability of ER and ERß to this site. The oligonucleotides were end-labeled with32P-dATP through the use of T4 polynucleotide kinase, adjusted to 50 000 cpm/0.25 ng/µL, and 1 µL/19-µL sample was added. After additional incubation for 5 minutes at room temperature, the samples were directly loaded on a pre-run 6.0% polyacrylamide gel with 5% glycerol and 5% Ficoll (MW 400 000; Sigma) in 0.5x TBE (0.5x: 0.045 mol/L Tris/borate, 0.002 mL EDTA) and run at 20 mA for 2 hours. Gels were vacuum-dried and developed using a Cyclone Storage Phosphor Screen (Packard Bioscience) or Kodak BioMax MR films (Fisher Scientific). Densitometric quantitation of the autoradiograms was performed using the Ultra-Violet Products imaging system and Labworks software.
Statistical Analysis
Statistical analyses of data presented included ANOVA and linear regression as indicated and were performed by using SPSS 11.0. When ANOVA showed a statistically significant difference between treatment groups (P<0.05), we then used Scheffe multiple comparison procedure to determine which pairs of treatment groups were significantly different. Data are reported as the mean±SEM unless otherwise indicated. Experiments designed to test the activity of ER against ERß on PAI-1 promoter reporters were performed independently 3 times in triplicate sets.
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Results |
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ER
and ERß Influence PAI-1 Promoter Activity Differentially in Endothelial CellsA schematic representation of the proximal upstream regulatory region of the human PAI-1 gene is shown in Figure 1A. Numerous enhancer/repressor elements have been identified, including glucose response sites,35 molecular clock responsive elements,39 and a glucocorticoid/mineralcorticoid element at –64 bp.41 To examine the potential role of ERs in regulating the expression of PAI-1 in vascular endothelial cells, plasmid constructs expression ER
, ERß, or pcDNA3.1 (empty vector) were cotransfected with a plasmid containing various PAI-1 promoter 5' deletions or mutations at selected sites (Figure 1B). Western blot analysis of nuclear extracts from transfected cells confirmed the expression of both pER and pERß constructs (Figure 1C). First, we characterized the effects of ER and ERß on activation of the –3.4kbp PAI-1 construct. There was evidence of endogenous expression of ER in BAEC transfected with the empty vector, as previously described.40 Cotransfection of –3.4kbp PAI-1 and pER in BAEC treated with estrogen (E2 100 nmol/L; Figure 2) increased PAI-1 promoter activity compared with empty vector (1.2±0.2 versus 4.2±0.6; P=0.003, by ANOVA with Scheffe test for multiple comparisons). The mean of the difference between the effects of pER and the empty vector is 1.2 (95% CI, 0.4 to 1.9). In contrast, cotransfection of –3.4kbp PAI-1 and pERß in BAEC had little or no effect on PAI-1 promoter activity compared with empty vector (1.2±0.2 versus 0.6±0.1; P=0.783, by ANOVA; Figure 2). We also investigated the effects of co-expressing ER and ERß in BAEC with –3.4kbp PAI-1, because pER and pERß exert differential effects on PAI-1 promoter activity when transfected independently. When pER and pERß are transfected together, in equimolar concentrations, ERß exerts a dominant repressive influence over the ability of ER to elicit promoter activation (4.2±0.6 versus 0.5±0.5, P=0.003, by ANOVA with a difference of means equal to 1.7 (95% CI, 0.9 to 2.4) by Scheffe test for multiple comparisons; Figure 2).
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Coordinate Regulation of PAI-1 Promoter Activity by ER and ERß
Because cooperative and inhibitory interactions have been observed between ER and ERß, we next investigated the possibility that endothelial ERs may interact in either an additive or dominant-negative manner. Cotransfection of binary mixtures of ER and ERß plasmids, each of a constant total mass but varied with respect to ER subtype stoichiometry, revealed the modulatory effects of ERß on PAI-1 promoter activity. Linear regression analysis demonstrated significant differences between the slopes of the regression lines generated for ER (17.4±1.4; 95% CI, 14.5 to 20.2) versus either ERß (7.0±1.1; 95% CI, 4.8 to 9.3) or ER plus ERß (0.5±0.5; 95% CI, –0.5 to 1.4; Figure 3A and B). The slopes of the regression lines of ERß versus ER plus ERß were not statistically significant. At high concentrations (>0.2 µg), ERß increased the activity of the PAI-1 promoter. The overall effect of increasing concentrations of ERß alone was to increase the activity of the PAI-1 promoter, albeit with less efficacy than ER. Therefore, the extent to which PAI-1 promoter activity was induced by ER in the presence of ERß depended on the stoichiometric ratio of the 2 ER subtypes, and the effect may be either to increase or to decrease the PAI-1 promoter activity.
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) and ERß plasmid (
) (A). A series of binary mixtures of ER
) reveals the modulatory effects of ERß on ER
An ERE Is Required for ER-Dependent Activation of the –0.8kbp PAI-1 Promoter Fragment
A search for classical EREs within a –0.8kbp PAI-1 promoter fragment revealed the presence of a sequence elements bearing significant similarity to a consensus ERE at positions –897 and –427 of the PAI-1 promoter (Figure 1A). Truncation of the PAI-1 promoter construct from 3.4kbp to 0.8kbp did not reduce the response to ER or ERß, so our efforts focused on the putative ERE at –427. To test the possibility that this element is involved in the induction of the PAI-1 promoter fragment by ER or the repression by ERß, the putative 3' half-site was altered by site-directed mutagenesis. Transfections of either mutant or wild-type promoters with ER demonstrated that the half-site mutation is sufficient to abolish the ER-dependent stimulation of the –0.8kbp PAI-1 luciferase activity (4.0±0.7 versus 1.0±0.1; P=0.008, by ANOVA; Figure 4A). In contrast, the ability of ERß to repress the activity of this promoter construct was not influenced by this targeted mutation (Figure 4B).
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In Vitro Transcribed ER and ERß Are Equally Capable of Binding the ERE Consensus Sequence
The ability of both ER and ERß to bind to the classic ERE consensus sequence in the PAI-1 promoter was investigated by EMSA. Both ER and ERß were probed for their ability to interact in vitro with this sequence using a DNA probe that contained the putative ERE (designated PAI-1 ERE). ERs incubated with 32P-labeled DNA probe PAI-1ERE resulted in the formation of a single DNA–protein complex (Figure 5). The intensity of this band was reduced, but not completely eliminated, in the presence of increasing concentrations of unlabeled ERE. This complex represents a specific DNA/protein interaction between ER and the PAI-1 ERE oligonucleotide. In addition, the intensity of this complex was not reduced when competed with unlabeled mutant ERE, which contained a mismatch base in the ERE (data not shown). ERß produced a similar pattern of specificity (Figure 5).
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ERß Increases PAI-1 Promoter Activity With Antiestrogens
Next, we examined the effects of estrogen and the selective ER modulator tamoxifen on transcriptional activation of the –3.4kbp PAI-1 promoter. In contrast to the data obtained with 17-ß estradiol (E2), ER failed to activate the PAI-1 promoter after treatment with low-dose (1.0 µmol/L) tamoxifen. Tamoxifen had little or no effect on the ERß-mediated suppression of PAI-1 promoter activity. There was, however, a modest but statistically significant enhancement of PAI-1 promoter activity in the cells transfected with equivalent masses of ER plus ERß treated with tamoxifen (0.9±0.1 versus 2.1±0.1, P<0.01 by ANOVA). The activation of PAI-1 by equivalent ER plus ERß and tamoxifen was comparable with that produced by ER plus estrogen (Figure 6). However, at a higher dose, tamoxifen (10 µmol/L) decreased the effects of both ER and ERß individually, as well as ER plus ERß together (data not shown).
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ER-Dependent Activation and ERß-Dependent Repression of the PAI-1 Promoter Does Not Require Intact AP-1 Sites
We performed additional experiments to determine whether the observed effects of ER and ERß on the PAI-1 promoter activity are AP1 site-dependent or ERE-dependent. Two AP1-like sites in the PAI-1 proximal promoter region are known to be critical for the serum-mediated activity of the PAI-1 promoter.42 Mutations of either the proximal AP1 site (P-box) or the distal AP1 site (D-box) in the proximal PAI-1 promoter markedly diminished PAI-1 promoter activity both in the presence and absence of ectopically expressed ER or ERß in transfected BAEC (Figure 7), but this reduction was not significant (P=0.09 by ANOVA). The relative ability of estrogen (E2) to induce PAI-1 promoter activity in the presence of ER is also diminished in the D-box mutant construct. However, neither the D-box nor the P-box mutations completely eliminated the ER-dependent activation or ERß-dependent suppression of the –3.4kbp PAI-1 promoter construct in BAEC (Figure 7).
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Discussion |
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ER
and ERß exert differential effects on the transcriptional activity of the human PAI-1 promoter in transient transfection studies using cultured endothelial cells. Our experiments demonstrate that ER increases the promoter activity of PAI-1 promoter fragments in response to estrogen. ER interacts with at least 1 ERE located at position –422 in the proximal PAI-1 promoter to elicit this estrogen-dependent activation of the PAI-1 promoter. In contrast, ERß fails to induce, and in fact may suppress, the activity of PAI-1 promoter constructs tested in BAECs through an estrogen-independent mechanism. Furthermore, ERß exerts a dominant-negative effect on ER to blunt the estrogen-dependent activation of the PAI-1 promoter in BAECs. Based on these findings, we speculate that the balance of ER and ERß in vascular tissue modulates vascular PAI-1 production via estrogen-dependent and estrogen-independent mechanisms.
The observations reported here are consistent with recent studies that show negative regulation of gene activity by ERß in promoters usually activated by ER.29,33 The effects of ERß on PAI-1 promoter activity are not modified by any of the promoter element mutations tested. This effect is not caused by competitive effects of DNA effector promoter with the target PAI-1 promoter because the ER construct is under the control of an identical cytomegalovirus-derived promoter. Thus, it seems likely that ERß uses a cellular mechanism that is distinct from the ER pathway to influence PAI-1 promoter activity. We propose that the unique functional properties of ERß revealed in our experiments may be explained by an additional element in the PAI-1 promoter that is involved in mediating the estrogen-independent activation of the PAI-1 promoter via ERß. Alternatively, ERß may indirectly influence PAI-1 promoter activity by signaling through a membrane linked-receptor system as recently reported.13,23,43
To further explore the role of the ERs in modulating the PAI-1 promoter activity in BAECs, we investigated the roles of 2 AP1-like promoter elements, the P-box and D-box, in mediating the ER responses at the PAI-1 promoter.42 Mutation of the P-box element severely impaired the serum-responsiveness of the PAI-1 promoter but did not appreciably modulate the ER or ERß actions. Similarly, mutation of the D-box element significantly reduced serum-dependent PAI-1 promoter activity without radically modulating the ERß response. The D-box mutant construct exhibited a reduced ER response, indicating a potential interaction between ER and the D-box. The relative inability of either mutant site to abolish ER responses argues that another element, such as an alternative ERE, may be required to mediate the estrogen-dependent response. To investigate the requirement for an ERE-type promoter element in mediating ER actions at the PAI-1 promoter, we identified and mutated a candidate ERE (–427) in the –0.8kbp PAI-1 promoter construct. This experiment illustrates the importance of this nucleic acid motif in conferring ER responsiveness. These results do not exclude the possibility that other ERE-type elements contribute to promoter responsiveness in the context of larger PAI-1 promoter fragments.
In this study, estrogen treatment failed to induce activity of the PAI-1 promoter via ERß or the mixture of ER plus ERß. In contrast, tamoxifen induced PAI-1 activity with the combination of ER plus ERß. The variability in ligand preferences for ER and ERß activation of the PAI-1 promoter are consistent with the previously observed preferential ligand binding at the AP-1–regulated cyclin D1 and collagenase promoters.33,44 We therefore conclude that the effect of estrogen on endothelial gene expression represents the combination of both synergistic and antagonistic actions by ER and ERß. Similarly, the observations reported in this study may reflect a direct interaction between these receptors independent of binding regulatory elements in the PAI-1 promoter.
Although the current in vitro model system is limited by the use of a single endothelial cell type removed from physiological conditions, this methodology allows one to examine the discrete effects of estrogen on PAI-1 promoter activity through 2 distinct receptors, both individually and in combination. Clearly, the native human PAI-1 promoter provides a convenient and physiological construct for testing the ER specificity of selective ER modulators. Further studies exploring the function of ER and ERß in a human endothelial cell system may be useful in resolving the mechanisms that underlie their differential effects on PAI-1 promoter activity in BAECs. As more selective ER modulators become available, it seems likely that studies using receptor-specific quantitative measurements will facilitate a more robust appreciation for the potential contribution of each receptor to a vascular protective effect of estrogen.
These findings may be interpreted to suggest that the reduction in plasma PAI-1 levels observed after treatment of postmenopausal women with hormone replacement therapy likely reflects local ERß-dependent mechanisms in the vasculature.4,11 An alternative explanation is that an ER cofactor that potentiates negative regulatory activity of ER on the PAI-1 promoter exists but was not present in the endothelial cells studied. Further investigation into the regulation of PAI-1 via ER and ERß will likely contribute to our understanding of the molecular mechanisms of estrogen on the vasculature and how estrogen impacts on the risk of thrombosis and cardiovascular disease.
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Acknowledgments |
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The work was supported by National Institutes of Health grants HL65192, HL51387, and T32-HL007411 (to D.E.V.). We thank Dan Byrne for his expert advice on the statistical methods used in this study.
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Footnotes |
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*These authors contributed equally to this work.
Original received July 9, 2001; resubmission received March 11, 2004; revised resubmission received June 1, 2004; accepted June 11, 2004.
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References |
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