Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2004;95:269-275
Published online before print June 24, 2004, doi: 10.1161/01.RES.0000136521.70093.f1
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
95/3/269    most recent
01.RES.0000136521.70093.f1v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, L. H.
Right arrow Articles by Vaughan, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, L. H.
Right arrow Articles by Vaughan, D. E.
Related Collections
Right arrow Endothelium/vascular type/nitric oxide
Right arrow Gene regulation
(Circulation Research. 2004;95:269.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Differential and Opposing Regulation of PAI-1 Promoter Activity by Estrogen Receptor {alpha} and Estrogen Receptor ß in Endothelial Cells

Layton Harris Smith*, Stephen R. Coats*, Hao Qin, Matthew S. Petrie, Joseph W. Covington, Ming Su, Mesut Eren, Douglas E. Vaughan

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha} and ERß) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ER{alpha} 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{alpha}. Mutation of a putative estrogen response element (ERE) located at position –427 in the proximal promoter abolished the ER{alpha} action without influencing the suppressive effects of ERß. Mutation of either AP1-like site did not eliminate the ER{alpha} 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{alpha} and ERß exert differential effects on the PAI-1 promoter activity in transfected BAECs. ER{alpha} 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{alpha} and ERß.


Key Words: estrogen receptors • plasminogen activator inhibitor-1 promoter • estrogen response element • bovine aortic endothelial cell


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha}, and ERß, function as ligand-dependent transcription factors.22,23 Although ER{alpha} 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{alpha} at subsaturating concentrations of ligand but has no effect on ER{alpha}-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{alpha} 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{alpha} but inhibits AP-1–dependent transcription via ERß,33 suggesting that 17ß-estradiol regulates ER{alpha}-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{alpha} and ERß in transactivating the human PAI-1 promoter. We also show that ER{alpha} 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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha}, was generated by ligating a human ER{alpha} 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{alpha} (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{alpha} 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{alpha} 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{alpha} against ERß on PAI-1 promoter reporters were performed independently 3 times in triplicate sets.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
ER{alpha} and ERß Influence PAI-1 Promoter Activity Differentially in Endothelial Cells
A 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{alpha}, 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{alpha} and pERß constructs (Figure 1C). First, we characterized the effects of ER{alpha} and ERß on activation of the –3.4kbp PAI-1 construct. There was evidence of endogenous expression of ER{alpha} in BAEC transfected with the empty vector, as previously described.40 Cotransfection of –3.4kbp PAI-1 and pER{alpha} 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{alpha} 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{alpha} and ERß in BAEC with –3.4kbp PAI-1, because pER{alpha} and pERß exert differential effects on PAI-1 promoter activity when transfected independently. When pER{alpha} and pERß are transfected together, in equimolar concentrations, ERß exerts a dominant repressive influence over the ability of ER{alpha} 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).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. A, Schematic representation showing the single nucleotide polymorphism and enhancer elements in the proximal PAI-1 promoter. ERE indicates putative estrogen response element; TGF-ß, transforming growth factor-ß;45 VLDL, very-low-density lipoprotein;46 GRE, glucocorticoid response element;41 4G/5G, common single nucleotide polymorphism.47 B, Plasmid PAI-1 promoter reporter constructs used in this study. Native nucleotide sequences of the promoter are indicated in normal type, mutated nucleotide sequences are indicated in bold type. Position of the luciferase coding region is indicated. C, Western blot of ER subtypes in transfected BAECs.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 2. Differential activity of ER{alpha} and ERß at the –3.4kbp PAI-1 promoter in BAEC. BAEC were transfected with the indicated plasmid constructs as described in "Materials and Methods" and treated for 48 hours in the presence of vehicle (0.1% ethanol) or E2 (100 nmol/L). Luciferase activities from triplicate samples are reported as relative induction.

Coordinate Regulation of PAI-1 Promoter Activity by ER{alpha} and ERß
Because cooperative and inhibitory interactions have been observed between ER{alpha} 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{alpha} 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{alpha} (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{alpha} 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{alpha} 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{alpha}. Therefore, the extent to which PAI-1 promoter activity was induced by ER{alpha} 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.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Coordinate regulation of PAI-1 promoter activity by ER{alpha} and ERß. The ordinate displays mean (±SEM) fold induction of PAI-1 promoter activity after cotransfection of a constant mass of PAI-1 plasmid with an increasing mass of ER{alpha} plasmid from 0.0 to 0.4 µg per well ({blacktriangleup}) and ERß plasmid ({circ}) (A). A series of binary mixtures of ER{alpha} and ERß plasmids ({blacksquare}) reveals the modulatory effects of ERß on ER{alpha} mediated induction of the PAI-1 promoter (B). Linear regression lines demonstrate trends in the data (regression line: solid line; 95% confidence intervals, dashed lines).

An ERE Is Required for ER{alpha}-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{alpha} 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{alpha} 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{alpha} demonstrated that the half-site mutation is sufficient to abolish the ER{alpha}-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).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. BAEC were transfected with the indicated constructs for 48 hours in the presence of vehicle (0.1% ethanol) or E2 (100 nmol/L). Luciferase activities from triplicate samples are reported as relative induction units. A, Effect of the mutant ERE half-site on ER{alpha}-dependent PAI-1 promoter activation. B, Effect of the mutant ERE half-site on ERß-dependent promoter activation.

In Vitro Transcribed ER{alpha} and ERß Are Equally Capable of Binding the ERE Consensus Sequence
The ability of both ER{alpha} and ERß to bind to the classic ERE consensus sequence in the PAI-1 promoter was investigated by EMSA. Both ER{alpha} 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).



View larger version (62K):
[in this window]
[in a new window]
  		Figure 5. Gel mobility shift assay, performed as described in Materials and Methods. Labeled probe was incubated with in vitro-translated ER{alpha} and ERß proteins in the absence or presence of cold competitor, as indicated at top. Upper bands represent bound probe and lower bands represent unbound probe.

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{alpha} 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{alpha} 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{alpha} plus ERß and tamoxifen was comparable with that produced by ER{alpha} plus estrogen (Figure 6). However, at a higher dose, tamoxifen (10 µmol/L) decreased the effects of both ER{alpha} and ERß individually, as well as ER{alpha} plus ERß together (data not shown).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 6. Effects of tamoxifen and ER subtypes at the –3.4kbp PAI-1 promoter in BAEC. Cells were transfected with the indicated plasmids and treated for 48 hours in the presence of vehicle (0.1% ethanol), E2 (0.1 µmol/L), or tamoxifen (1.0 µmol/L). Luciferase activities are reported as mean (±SEM) fold induction of control.

ER{alpha}-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{alpha} 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{alpha} 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{alpha} is also diminished in the D-box mutant construct. However, neither the D-box nor the P-box mutations completely eliminated the ER{alpha}-dependent activation or ERß-dependent suppression of the –3.4kbp PAI-1 promoter construct in BAEC (Figure 7).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 7. Influence of mutated AP1-like sites on PAI-1 promoter activity in the presence of ER{alpha} and ERß. BAEC were transfected with the indicated plasmid constructs and treated for 48 hours in the presence of vehicle (0.1% ethanol) or estrogen (100 nmol/L). Global, serum-dependent –3.4kbp PAI-1 promoter activity depends on P-box and D-box function.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
ER{alpha} 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{alpha} increases the promoter activity of PAI-1 promoter fragments in response to estrogen. ER{alpha} 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{alpha} to blunt the estrogen-dependent activation of the PAI-1 promoter in BAECs. Based on these findings, we speculate that the balance of ER{alpha} 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{alpha}.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{alpha} 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{alpha} 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{alpha} 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{alpha} response, indicating a potential interaction between ER{alpha} 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{alpha} 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{alpha} plus ERß. In contrast, tamoxifen induced PAI-1 activity with the combination of ER{alpha} plus ERß. The variability in ligand preferences for ER{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha} 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.


*    Acknowledgments
 
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.


*    Footnotes
 
*These authors contributed equally to this work. Back

Original received July 9, 2001; resubmission received March 11, 2004; revised resubmission received June 1, 2004; accepted June 11, 2004.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1 and coronary artery disease. N Engl J Med. 2000; 342: 1792–1801.[Free Full Text]
  2. Hamsten A, de Faire U, Walldius G, Dahlen G, Szamosi A, Landou C, Blomback M, Wiman B. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 1987; 2: 3–9.[CrossRef][Medline] [Order article via Infotrieve]
  3. Blum A, Hathaway L, Mincemoyer R, Csako G, Waclawiw MA, Panza JA, Cannon RO,3rd, Koh KK. Effects of hormone-replacement therapy on fibrinolysis in postmenopausal women. Circulation. 1999; 100: 1851–1857.[Abstract/Free Full Text]
  4. Brown NJ, Abbas A, Byrne D, Schoenhard JA, Vaughan DE. Comparative effects of estrogen and angiotensin-converting enzyme inhibition on plasminogen activator inhibitor-1 in healthy postmenopausal women. Circulation. 2002; 105: 304–309.[Abstract/Free Full Text]
  5. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Medicine. 1991; 20: 47–63.
  6. Grodstein F. Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N Engl J Med. 1996; 335: 453–461.[Abstract/Free Full Text]
  7. Manson JE HJ, Johnson KC, Rossouw JE, Assaf AR, Lasser NL, Trevisan M, Black HR, Heckbert SR, Detrano R, Strickland OL, Wong ND, Crouse JR, Stein E, Cushman M; Women’s Health Initiative Investigators. Estrogen plus progestin and the risk of coronary heart disease. N Engl J Med. 2003; 394: 523–534.
  8. Herrington D, Bittner V, Blumenthal R, Davidson M, Hlatky M, Hsia J, Hulley S, Herd A, Khan S, Newby LK, Waters D, Herrington DM. Statin therapy, cardiovascular events, and total mortality in the Heart and Estrogen/Progestin Replacement Study (HERS). Circulation. 2002; 105: 2962–2967.[Abstract/Free Full Text]
  9. Hulley S. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA. 1998.
  10. Rossouw JE. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. JAMA. 2002; 288: 321–333.[Abstract/Free Full Text]
  11. Joswig M, Hach-Wunderle V, von Holst T, Nawroth PP. Postmenopausal hormone replacement therapy and the vascular wall: epidemiology and clinical studies. Vasa. 2000; 29: 243–251.[CrossRef][Medline] [Order article via Infotrieve]
  12. Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. Am J Cardiol. 2002; 89: 12E–17E;discussion 17E–18E.
  13. Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, Hidaka H, Iguchi A. Estrogen increases endothelial nitric oxide by a receptor-mediated system. Biochem Biophys Res Com. 1995; 214: 847–855.[CrossRef][Medline] [Order article via Infotrieve]
  14. de Valk-de Roo GW, Stehouwer CD, Meijer P, Mijatovic V, Kluft C, Kenemans P, Cohen F, Watts S, Netelenbos C. Both raloxifene and estrogen reduce major cardiovascular risk factors in healthy postmenopausal women: A 2-year, placebo-controlled study. Arterioscler Thromb Vasc Biol. 1999; 19: 2993–3000.[Abstract/Free Full Text]
  15. Giltay EJ, Gooren LJ, Emeis JJ, Kooistra T, Stehouwer CD. Oral, but not transdermal, administration of estrogens lowers tissue-type plasminogen activator levels in humans without affecting endothelial synthesis. Arterioscler Thromb Vasc Biol. 2000; 20: 1396–1403.[Abstract/Free Full Text]
  16. Mendelsohn ME, Karas RH. Protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999; 340: 1801–1811.[Free Full Text]
  17. Grunenwald D, Pujol JL, Girard P, Dujon A, Brouchet L, Brichon PY, Westeel V, Le Chevalier T. Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta. Cancer. 2001; 91: 2394–2400.[CrossRef][Medline] [Order article via Infotrieve]
  18. Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR, Jr., Lubahn DB, O’Donnell TF, Jr., Korach KS, Mendelsohn ME. Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat Med. 1997; 3: 545–548.[CrossRef][Medline] [Order article via Infotrieve]
  19. Karas RH, Hodgin JB, Kwoun M, Krege JH, Aronovitz M, Mackey W, Gustafsson JA, Korach KS, Smithies O, Mendelsohn ME. Estrogen inhibits the vascular injury response in estrogen receptor beta-deficient female mice. Proc Natl Acad Sci U S A. 1999; 96: 15133–15136.[Abstract/Free Full Text]
  20. Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, Price RH, Jr, Pestell RG, Kushner PJ, Nilsson S. Mechanisms of estrogen action. J Biol Chem. 2002; 277: 24353–24360.[Abstract/Free Full Text]
  21. Ekblad E, Gustafsson JA, Nilsson BO, Bracamonte MP. Vascular effects of estrogens: arterial protection versus venous thrombotic risk. J Endocrinol. 2001; 171: 417–423.[Abstract]
  22. Kumar V, Green S, Staub A, Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986; 5: 2231–2236.[Medline] [Order article via Infotrieve]
  23. Razandi M, Pedram A, Greene GL, Levin ER. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol Endocrinol. 1999; 13: 307–319.[Abstract/Free Full Text]
  24. Albanese C, Anderson CM, Hilly K, Webb P, Uht RM, Price RH Jr, Pestell RG, Kushner PJ, Nillson S. Mechanisms of estrogen action. J Phys Rev. 2001; 81: 1535–1565.
  25. Deleted in proof.
  26. Katzenellenbogen BS, Choi I, Delage-Mourroux R, Ediger TR, Martini PG, Montano M, Sun J, Weis K, Katzenellenbogen JA. Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology. J Steroid Biochem Mol Biol. 2000; 74: 279–285.[CrossRef][Medline] [Order article via Infotrieve]
  27. Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor beta acts as a dominant regulator of estrogen signaling. Oncogene. 2000; 19: 4970–4978.[CrossRef][Medline] [Order article via Infotrieve]
  28. Kumar V, Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988; 55: 145–156.[CrossRef][Medline] [Order article via Infotrieve]
  29. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E, Katzenellenbogen BS, Enmark E, Gustafsson J-A, Nilsson S, Kushner PJ. The Estrogen Receptor Enhances AP-1 Activity by Two Distinct Mechanisms with Different Requirements for Receptor Transactivation Functions. Mol Endocrinol. 1999; 13: 1672–1685.[Abstract/Free Full Text]
  30. Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson J-A, Safe S. Ligand-, Cell-, and Estrogen Receptor Subtype (alpha /beta)-dependent Activation at GC-rich (Sp1) Promoter Elements. J Biol Chem. 2000; 275: 5379–5387.[Abstract/Free Full Text]
  31. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P. Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol. 2000; 74: 311–317.[CrossRef][Medline] [Order article via Infotrieve]
  32. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology. 1997; 138: 863–870.[Abstract/Free Full Text]
  33. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-A, Kushner PJ, Scanlan TS. Differential Ligand Activation of Estrogen Receptors ER{alpha} and ER at AP1 Sites. Science. 1997; 277: 1508–1510.[Abstract/Free Full Text]
  34. Vaughan DE, Lazos SA, Tong K. Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells. A potential link between the renin-angiotensin system and thrombosis. J Clin Invest. 1995; 95: 995–1001.[Medline] [Order article via Infotrieve]
  35. Chen YQ, Su M, Walia RR, Hao Q, Covington JW, Vaughan DE. Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells. J Biol Chem. 1998; 273: 8225–8231.[Abstract/Free Full Text]
  36. Needleman S, Wunsch C. A general method applicable to the seach for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970; 48: 443–453.[CrossRef][Medline] [Order article via Infotrieve]
  37. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol. 1997; 11: 353–365.[Abstract/Free Full Text]
  38. Campbell LH, Schmidt MG, Arrigo SJ, Gatlin J. Direct-rapid (DR) mutagenesis of large plasmids using PCR. Biotechniques. 1995; 19: 559–564.[Medline] [Order article via Infotrieve]
  39. Schoenhard JA, Smith LH, Painter CA, Eren M, Johnson CH, Vaughan DE. Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2. J Mol Cell Cardiol. 2003; 35: 473–481.[CrossRef][Medline] [Order article via Infotrieve]
  40. Venkov CD, Rankin AB, Vaughan DE. Identification of authentic estrogen receptor in cultured endothelial cells. A potential mechanism for steroid hormone regulation of endothelial function. Circulation. 1996; 94: 727–733.[Abstract/Free Full Text]
  41. Brown NJ, Kim KS, Chen YQ, Blevins LS, Nadeau JH, Meranze SG, Vaughan DE. Synergistic effect of adrenal steroids and angiotensin II on plasminogen activator inhibitor-1 production. J Clin Endocrinol Metabol. 2000; 85: 336–344.[Abstract/Free Full Text]
  42. Knudsen H, Olesen T, Riccio A, Ungaro P, Christensen L, Andreasen PA. A common response element mediates differential effects of phorbol esters and forskolin on type-1 plasminogen activator inhibitor gene expression in human breast carcinoma cells. Eur J Biochem. 1994; 220: 63–74.[Medline] [Order article via Infotrieve]
  43. Heaton JH, Tillmann-Bogush M, Leff NS, Gelehrter TD. Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences. J Biol Chem. 1998; 273: 14261–14268.[Abstract/Free Full Text]
  44. Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, Price RH Jr, Pestell RG, Kushner PJOpposing action of estrogen receptors alpha and beta on cyclin D1 gene expression. J Biol Chem. 2002; 277: 24353–24360.[Abstract/Free Full Text]
  45. Keeton MR, Curriden SA, van Zonneveld AJ, Loskutoff DJ. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta. J Biol Chem. 1991; 266: 23048–23052.[Abstract/Free Full Text]
  46. Dichtl W, Ares MP, Stollenwerk M, Giachelli CM, Scatena M, Hamsten A, Eriksson P, Nilsson J. In vivo stimulation of vascular plasminogen activator inhibitor-1 production by very low-density lipoprotein involves transcription factor binding to a VLDL-responsive element. Thromb Haemost. 2000; 84: 706–711.[Medline] [Order article via Infotrieve]
  47. Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S, Henney AM. The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells. J Biol Chem. 1993; 268: 10739–10745.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J. M. Wang, R. W. Irwin, and R. D. Brinton
Activation of estrogen receptor {alpha} increases and estrogen receptor beta decreases apolipoprotein E expression in hippocampus in vitro and in vivo
PNAS, November 7, 2006; 103(45): 16983 - 16988.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise
Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies
Endocr. Rev., October 1, 2006; 27(6): 575 - 605.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella
Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease
Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2192 - 2199.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Urata, Y. Ihara, H. Murata, S. Goto, T. Koji, J. Yodoi, S. Inoue, and T. Kondo
17beta-Estradiol Protects against Oxidative Stress-induced Cell Death through the Glutathione/Glutaredoxin-dependent Redox Regulation of Akt in Myocardiac H9c2 Cells
J. Biol. Chem., May 12, 2006; 281(19): 13092 - 13102.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
L. H. Smith, M. S. Petrie, J. D. Morrow, J. A. Oates, and D. E. Vaughan
The sterol response element binding protein regulates cyclooxygenase-2 gene expression in endothelial cells
J. Lipid Res., May 1, 2005; 46(5): 862 - 871.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
N. Madamanchi, X. Liu, and M. S. Runge
A New Slice of Pie?: Estrogen Regulation of Plasminogen Activator Inhibitor-1
Circ. Res., August 6, 2004; 95(3): 228 - 229.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
95/3/269    most recent
01.RES.0000136521.70093.f1v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smith, L. H.
Right arrow Articles by Vaughan, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, L. H.
Right arrow Articles by Vaughan, D. E.
Related Collections
Right arrow Endothelium/vascular type/nitric oxide
Right arrow Gene regulation

Circulation Research Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2004 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.