Specific Regulation of Lipocalin-Type Prostaglandin D Synthase in Mouse Heart by Estrogen Receptor ß
Michio Otsuki,
Hui Gao,
Karin Dahlman-Wright,
Claes Ohlsson,
Naomi Eguchi,
Yoshihiro Urade and
Jan-Åke Gustafsson
Department of Biosciences at Novum (M.O., H.G., K.D.-W., J.-A.G.), Karolinska Institutet, Huddinge SE-14157, Sweden; Division of Endocrinology (C.O.), Department of Internal Medicine, Sahlgrenska University Hospital, Göteborg SE-41345, Sweden; and Department of Molecular Behavioral Biology (N.E., Y.U.), Osaka Bioscience Institute, Osaka 565-0874, Japan
Address all correspondence and requests for reprints to: Dr. Michio Otsuki, Department of Biosciences at Novum, Karolinska Institutet Huddinge SE-14157, Sweden. E-mail michio.otsuki{at}biosci.ki.se.
 |
ABSTRACT
|
---|
Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERß in vivo. In further support of ERß-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERß, but not by ER
. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.
 |
INTRODUCTION
|
---|
EPIDEMIOLOGICAL AND OBSERVATIONAL studies have suggested that estrogens have beneficial effects on the cardiovascular system. For example, the incidence of hypertension and cardiovascular disease is significantly lower in premenopausal women than in men, whereas after the menopause, this difference disappears (1, 2, 3). This indicates that estrogens may play an important role in the prevention of cardiovascular disease in women. However, case control studies have failed to show any clear beneficial effects of estrogens with regard to primary and secondary prevention (4). The estrogen-progestin arm of the Womens Health Initiative trial (WHI) was stopped because of unacceptable risk-benefit profiles. The estrogen arm of this study is still on-going (5). Whether or not estrogen agonists and/or antagonists will find a place in the treatment of cardiovascular disease, they will remain important pharmaceutical compounds for other indications and, consequently, the molecular effects of estrogen on the cardiovascular system need to be characterized. The majority of studies have concentrated on the vascular effects of the hormone (6). However, whereas cardiac mass increases with age in apparently healthy women, it remains constant in men. Under increased cardiac loading conditions, such as hypertension or aortic stenosis, this difference between sexes is even more striking (7). Cardiac mass in a long-term hormone replacement therapy group is lower than in the control group after compensation for known determinants of left ventricular mass (8), and estrogen attenuates the development of pressure-overload hypertrophy in mice (9). These findings suggest that the heart is also an important target organ for estrogen.
The mechanisms of estrogen action are known to be mediated by two estrogen receptors, estrogen receptor
(ER
) and estrogen receptor ß (ERß) (10, 11, 12). The ERs act as ligand-activated nuclear transcription factors. The tissue distributions of the ERs are different. ER
has a broad expression pattern, whereas ERß has a more focused pattern with high levels in the ovary, prostate, epididymis, lung, heart, blood vessels, mammary gland, colon, stomach, hematopoietic system, and brain (13, 14, 15, 16, 17). Both ERs are expressed in cardiac myocytes and fibroblasts (18).
ER binds to specific sequences, called estrogen response elements (EREs), with high affinity and transactivates gene expression in response to estradiol.
ERß knockout mice, in which the gene for ERß has been disrupted, display an interesting phenotype in the cardiovascular system (abnormalities of ion channel function and sustained systolic and diastolic hypertension as they age) (19). This report suggests that ERß may play an important role in the heart. The details of the heart phenotype in ERß-deleted mice remain elusive.
Systematic studies of gene expression patterns using cDNA microarray technologies provide a powerful and unbiased approach to dissect molecular events in cells and tissues by comparing expression levels of thousands of genes at a time (20). Recently, cDNA microarray analysis was shown to be an efficient method to assess hormonal regulation of target genes and their patterns of expression (21).
In this report we have investigated the effect of long-term estrogen treatment in the heart using oligonucleotide-based DNA microarrays (Affymetrix Gene Chip Probe Array) and characterized the molecular regulation of a novel estrogen-regulated gene in the heart.
 |
RESULTS
|
---|
Effect of Long-Term Estrogen Treatment on Global Gene Expression in the Heart
At first, we investigated the effect of long-term estrogen treatment on global gene expression in the heart. Serum 17ß-estradiol levels were not detectable (<5 pg/ml) in the vehicle treatment group (n = 4) and were 112 ± 45 pg/ml in the estrogen treatment group (n = 4). These 17ß-estradiol levels in the estrogen treatment group are similar to levels in other reports using equivalent protocols (9, 22). We compared gene expression profiles for vehicle-treated mice and estrogen-treated mice. Using Affymetrix Gene Chip Probe Arrays, we identified 16 genes that had more than 77% concordance change in nine pair-wise comparisons and at least a 1.4-fold change. Seven genes were up-regulated by estrogen, and nine genes were down-regulated by estrogen (Table 1
).
View this table:
[in this window]
[in a new window]
|
Table 1. Effect of Long-Term Estrogen Treatment on Gene Expression in the Heart (Affymetrix GeneChip Probe Array Analysis)
|
|
We used Northern blot analysis to confirm the result of the DNA microarray analysis. Northern blot analysis showed that estrogen increased lipocalin-type prostaglandin D synthase (L-PGDS) mRNA levels (3.9-fold), whereas estrogen decreased
1, type III procollagen mRNA levels (1.7-fold) (Fig. 1
).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1. Effect of Long-Term Estrogen Treatment on L-PGDS and 1, Type III Procollagen mRNA Expression in the Heart
Mice were treated with vehicle (V) or estrogen (E2, 100 µg/kg), respectively, for 3 wk. Total RNA was isolated from heart. Total RNA (15 µg) was separated by electrophoresis and transferred to nylon membranes, which were hybridized with 32P-labeled mouse L-PGDS, 1, type III procollagen, or GAPDH probe, respectively. The left panel (A) shows two representative experiments. The right panel (B) shows mean ± SD; L-PGDS, n = 3 per group; 1, type III procollagen, n = 6 per group. Data are normalized to GAPDH. *, P < 0.01 vs. respective vehicle treatment group.
|
|
Chronic Estrogen Treatment Effect of L-PGDS Protein Levels in the Heart
The following data presented in this report focus on a novel estrogen-regulated gene in the heart, L-PGDS, and characterize the molecular regulation of this gene by estrogen. To examine whether the increase in L-PGDS mRNA by estrogen was associated with an increase in the amount of the encoded protein, we performed Western blot analysis using a polyclonal anti-L-PGDS antibody. Whole-cell extracts from heart, treated with vehicle and estrogen for 3 wk, were used. As shown in Fig. 2
, estrogen treatment significantly increased PGDS protein levels (2.7-fold) after 3 wk of estrogen treatment. This demonstrates that changes in PGDS mRNA levels are reflected as changes in the corresponding protein.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2. Chronic Estrogen Treatment Effect of L-PGDS Protein Levels in the Heart
Mice were treated with vehicle (V) or estrogen (E2, 100 µg/kg) for 3 wk. Total cell lysates were extracted from hearts. Proteins (50 µg) were resolved by SDS-PAGE, blotted onto PVDF membranes, and then probed with an anti-L-PGDS polyclonal antibody. The top panel shows two representative experiments. The bottom panel shows mean ± SD; n = 7 per group. *, P < 0.01 vs. respective vehicle treatment group. A lysate from brain is used as positive control.
|
|
Reduction of Endogenous Estrogen Levels Suppresses L-PDGS Expression
Next we compared L-PGDS mRNA and protein expression between sham-operated mice and ovariectomized mice to investigate the effect of ovariectomy, and subsequent reduction in endogenous estrogen levels, on L-PGDS mRNA and protein expression. As expected, ovariectomized mice showed a significantly reduced uterine weight (35.0 ± 5.8 mg, n = 5) compared with sham-operated mice (108.0 ± 19.2 mg, n = 4, P < 0.01). As shown in Figs. 3
and 4
, L-PGDS mRNA and protein in heart from ovariectomized mice are significantly lower than in sham-operated mice (mRNA,
4.0-fold; protein,
4.5-fold). This experiment clearly demonstrates that L-PDGS mRNA and protein levels are influenced by estrogen levels within the physiological range.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3. Effect of Ovariectomy on L-PGDS mRNA Expression in the Heart
Mice were ovariectomized (OVX) or subjected to a sham operation (Sham). Total RNA was isolated from the heart. Total RNA (15 µg) was separated by electrophoresis and transferred to nylon membranes, which were hybridized with 32P-labeled mouse L-PGDS or GAPDH probes, respectively. The top panel (A) shows two representative experiments. The bottom panel (B) shows mean ± SD; n = 3 per group. Data are normalized to GAPDH. *, P < 0.01 vs. respective Sham group.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4. Effect of Ovariectomy on L-PGDS Protein Levels in the Heart
Mice were ovariectomized (OVX) or subjected to a sham operation (Sham). Total cell lysates were extracted from hearts. Proteins (50 µg) were resolved by SDS-PAGE, blotted onto PVDF membranes, and then probed with an anti-L-PGDS polyclonal antibody. The top panel shows two representative experiments. The bottom panel shows mean ± SD; n = 3 per group. **, P < 0.05 vs. respective Sham group. A lysate from brain is used as positive control.
|
|
L-PDGS Is Specifically Regulated via ERß in Vivo
Experiments using ER-disrupted mice were performed to elucidate the respective contribution of the two ERs in the induction of the L-PGDS gene in the heart. Northern blot analysis (Fig. 5
) showed that estrogen induced L-PGDS mRNA in ER
-disrupted mice (ERKO) but not in ERß-disrupted mice (BERKO) or ER
- and ß-disrupted mice (DERKO) mice. This shows that ERß plays an important role in estrogen-induced L-PGDS gene expression in the heart.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 5. Effect of Estrogen Treatment on L-PGDS mRNA Expression in the Heart of ER-Disrupted Mice
Mice were treated with vehicle (V) or estrogen (E2, 100 µg/kg) for 3 wk. Total RNA was isolated from the hearts. Total RNA (15 µg) was separated by electrophoresis and transferred to nylon membranes, which were hybridized with 32P-labeled mouse L-PGDS or GAPDH probe, respectively. The top panel shows one representative experiment. The bottom panel shows mean ± SD; WT, n = 3 per group; ERKO, n = 5 per group; BERKO, n = 5 per group; DERKO, n = 3 per group. Data are normalized to GAPDH. *, P < 0.01 vs. respective WT vehicle treatment group.
|
|
The L-PGDS Promoter Contains a Functional ERE That Is Specifically Activated by ERß
In Fig. 6
, we show that L-PGDS mRNA levels are already increased 1.7-fold after 6 h of estrogen treatment compared with vehicle treatment. This result suggests that L-PGDS is directly regulated by estrogen and prompted us to do the following studies on the L-PDGS promoter.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6. Effect of Short-Term Estrogen Treatment on L-PGDS mRNA Expression in the Heart
Mice were treated with vehicle (V) or estrogen (E2, 100 µg/kg) for 6 h. Total RNA was isolated from the hearts. Total RNA (15 µg) was separated by electrophoresis and transferred to nylon membranes, which were hybridized with 32P-labeled mouse L-PGDS or GAPDH probes, respectively. The left panel (A) shows two representative experiments. The bottom panel (B) shows mean ± SD; L-PGDS, n = 3 per group. Data are normalized to GAPDH. *, P < 0.01 vs. respective vehicle treatment group.
|
|
We searched the promoter region of L-PGDS (the 4-kb upstream region) for EREs, Sp1 sites, and activator protein 1 (AP-1) sites. We identified one ERE and one AP-1 site, respectively, in the approximately -4 kb to -3 kb upstream region. An approximately 1.0-kb fragment of the 5'-flanking region of the mouse L-PGDS gene (positions -4037/-3265), including these two response elements, was cloned and inserted upstream of a luciferase reporter gene in the pGL3 promoter vector (Fig. 7A
) to produce pGL3-L-PGDS (-4037/-3265). pGL3-L-PGDS (-4037/-3265) was transiently transfected into 293 cells together with ER
or ERß expression plasmids as indicated (Fig. 7B
). Coexpression of ERß in the presence of estradiol (10 nM) increased the reporter activity by 1.8-fold. On the contrary, coexpression of ER
did not activate the reporter plasmid (1.1-fold). Under similar experimental conditions, both ER
and ERß expression plasmids can activate a 2 x ERE-thymidine kinase Luc reporter plasmid (data not shown). These data show that the ERß-specific regulation of L-PDGS observed in vivo can be mimicked in an in vitro transient transfection system based on a defined fragment from the L-PDGS promoter.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7. ERß, But Not ER , Can Activate the L-PGDS Promoter
A, Schematic representation of the L-PGDS-luciferase reporter construct containing the mouse L-PGDS promoter region (-4037/-3265). B, 293 cells were transfected with the pGL3-L-PGDS (-4037/-3265) construct and ER or ERß expression plasmids, respectively. Cells were treated with ethanol or 10 nM 17ß-estradiol as indicated. The results are presented as fold induction compared with cells transfected with only the luciferase reporter and treated with ethanol. Values represent the mean ± SD of three independent experiments.
|
|
To examine which of the binding sites (ERE or AP-1 site) is important for the estrogen-induced L-PDGS expression, vectors including mutant versions of these sites (pGL3-L-PGDS EREm and pGL3-L-PGDS AP-1m) were constructed. Coexpression of ERß in the presence of estradiol (10 nM) increased the reporter activity by 1.6-fold with the pGL3-L-PGDS AP-1m construct (Fig. 8A
), but did not affect the reporter activity of the pGL3-L-PGDS EREm construct (Fig. 8B
). These results show that the ERE is the important element for ERß-mediated activation of pGL3-L-PGDS (-4037/-3265).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 8. The ERE Is Important for ERß-Mediated L-PGDS Promoter Activation
293 Cells were transfected with the pGL3-L-PGDS-AP-1m construct (A), the pGL3-L-PGDS-EREm (B) construct, and ER or ERß expression plasmids. Cells were treated with ethanol or 10 nM 17ß-estradiol as indicated. The results are presented as fold induction compared with cells transfected with only the luciferase reporter and treated with ethanol. Values represent the mean ± SD of three independent experiments.
|
|
To determine whether ERs bind to the ERE in L-PGDS promoter (L-PGDS ERE), EMSAs were performed with a double-stranded oligonucleotide containing the L-PGDS ERE. Figure 9
suggests that ERß bound the L-PGDS ERE with higher affinity than ER
(Fig. 9
, lanes 1 and 2). ER-L-PGDS ERE complexes were inhibited by an excess of unlabeled L-PGDS ERE oligonucleotide (Fig. 9
, lanes 3 and 4), but not by an excess of a nonspecific competitor oligonucleotide (Fig. 9
, lanes 5 and 6). Introduction of 2-bp substitutions created L-PGDS EREm, which did not bind ER
or ERß (Fig. 9
, lanes 7 and 8). Finally, lanes 11 and 12 show that the ERE from the Xenopus Vitellogenin A2 gene binds ER
with similar affinity as ERß. It is also apparent from these studies that the affinity for the Xenopus Vitellogenin A2 ERE is significantly higher than that for the L-PDGS ERE for both receptors.

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 9. ERß Binds the L-PGDS ERE with Higher Affinity than ER
The double-stranded probes, L-PGDS ERE, L-PGDS EREm, L-PGDS ERE (C to A), and Xenopus Vitellogenin A2 (X. Vite. A2) ERE are labeled with 32P and incubated with recombinant human ER (lanes 1, 3, 5, 7, 9, and 11) or ERß (lanes 2, 4, 6, 8, 10, and 12). The competitor L-PGDS ERE and a nonspecific competitor oligonucleotide (NS) were used in 100-fold excess (lanes 36). DNA-protein complexes were resolved on a nondenaturing acrylamide gel. The gel was dried and autoradiographed at -70 C.
|
|
Identification of a Nucleotide Position that Is Important for ERß-Selective Gene Activation and DNA Binding
To try to elucidate the molecular details of receptor selectivity, we compared the L-PGDS ERE and the Xenopus Vitellogenin A2 ERE (Fig. 10A
). One nucleotide (A to C) is altered in the L-PGDS ERE compared with Xenopus Vitellogenin A2 ERE. To investigate whether this nucleotide change plays an important role in ERß-specific transactivation and ERß-L-PGDS ERE binding, we made the pGL3-L-PGDS ERE (C to A) reporter plasmid for transfection experiments and the L-PGDS ERE (C to A) oligonucleotide, for EMSAs. Figure 9
(lanes 9 and 10) demonstrates that ER
and ERß bound to L-PGDS ERE (C to A) with higher affinity than the L-PGDS ERE. pGL3-L-PGDS ERE (C to A) was transiently transfected into 293 cells together with ER
or ERß expression plasmids as indicated (Fig. 10B
). Not only coexpression of ERß, but also that of ER
in the presence of estradiol (10 nM), increased the promoter activity by 2.6-fold (ERß) and 1.9-fold (ER
) in this construct. Specifically, ERß-induced pGL3-L-PGDS ERE (C to A) luciferase activity was higher than that achieved with the construct pGL3-L-PGDS (-4039/-3266). This result was concordant with the EMSAs. To determine whether the A to C change is sufficient to achieve receptor selectivity, we introduced the A to C change into the Vitellogenin A2 ERE. Coexpression of ER
in the presence of estradiol (10 nM) increased the reporter activity to the same level as that achieved with ERß in pGL3-Xenopus Vitellogenin A2 ERE, containing the wild-type (WT) Vitellogenin ERE. Importantly, ERß-induced pGL3-Xenopus Vitellogenin A2 ERE (A to C) reporter activity to a significantly higher level than ER
. These results demonstrate that a one-nucleotide (A to C) change in the consensus ERE contributes to ERß selective ERE induction. However, flanking sequences are likely to contribute to maximal selectivity.
 |
DISCUSSION
|
---|
Of 16 genes found to be regulated by estrogen in our study, using microarray technology, 15 have not been reported previously as estrogen target genes. Only
1, type I procollagen has previously been reported as an estrogen-regulated gene in the heart (23). Seven genes were up-regulated, whereas the remaining genes were down-regulated by estrogen. Half of the down-regulated genes encode extracellular matrix (ECM) proteins such as
1, type I procollagen,
1, type III procollagen, type XV procollagen, and secreted protein acidic and rich in cysteine. These ECM proteins are produced by cardiac fibroblasts. Cardiac fibroblasts, which comprise 60% of the total number of heart cells, may contribute to pathological structural changes in the heart by proliferating, depositing ECM proteins, and replacing tissue rich in myocytes with fibrotic tissue (24). It has been reported that estradiol inhibits cardiac fibroblast growth via an ER-independent pathway that involves local metabolism of estradiol to methoxyestradiols (25). The down-regulated thymosin ß 10, which is an actin monomer-sequestering protein (26), might play an important role in actin dynamics.
Next we focused on the molecular regulation of L-PGDS, which is one of the novel estrogen-regulated genes in the heart. Our data demonstrate that long-term (3 wk) estrogen treatment increased the expression of L-PGDS mRNA and protein in the heart. We also show that short-term (6 h) estrogen treatment increased L-PGDS mRNA and that ovariectomy reduces L-PGDS mRNA and protein. Our data suggest that physiological doses of estrogen may directly regulate L-PGDS gene expression. However, it cannot strictly be excluded that the acute and chronic effects of estrogen on L-PGDS expression occur via distinct mechanisms. We identified one ERE and one AP-1 site between -4 kb and -3 kb of the L-PDGS upstream region. Using L-PGDS promoter-luciferase reporter, we found that ERß, but not ER
, can activate this reporter, suggesting that this ERE plays an important role in the specific ERß-mediated activation of L-PDGS observed in vivo. Furthermore, DNA-binding studies suggest that ERß bound to the L-PDGS ERE with higher affinity than ER
. However, it is clear that the ERß selectivity for L-PDGS ERE is much more stringent in the transactivation assay and in the in vivo experiments than in the DNA-binding assay. Further studies are clearly needed to determine whether DNA binding is the sole determinant for receptor selectivity or whether differential cofactor recruitment, for example, contributes to the observed receptor selectivity. A comparison of this L-PGDS ERE with Xenopus Vitellogenin A2 ERE shows that the two elements are identical in the left half of palindrome, but differ at position 3 in the right half of the palindrome. Rabbit uteroglobin gene has the same ERE sequence as the L-PGDS ERE (27). It has been reported that the relative affinity of the rabbit uteroglobin ERE for ER
is lower than that of Xenopus Vitellogenin A2 ERE (27) and, similar to our studies, this ERE was not activated by transfected ER
(28). However, these studies did not include studies of receptor selectivity. Our studies (Fig. 10
, B and D) show that the A to C change contributes to receptor selectivity but that flanking sequences are probably important to achieve maximal selectivity. Further studies are required to fully understand the molecular mechanism of ERß/ER
-selective DNA binding and transactivation through the L-PGDS ERE.
Physiological effects of estrogen are mediated by ER
and ERß. The individual functions of the two ERs in the heart are poorly understood. However, BERKOs have abnormalities in ion channel function and sustained systolic and diastolic hypertension as they age (19). These data suggest that ERß has an important physiological role in heart function.
L-PGDS gene expression in the central nervous system is regulated by glucocorticoid and thyroid hormones (29, 30). Very recently, a report appeared showing estrogen regulation of L-PGDS in the central nervous system (31). Our results represent the first description of estrogen regulation of L-PGDS in the heart.
L-PGDS catalyzes the isomerization of prostaglandin H2, a common precursor of various prostanoids, to produce prostaglandin D2 (PGD2) (32). PGD2 is produced in a variety of tissues as a major prostanoid and has numerous physiological and pathophysiological functions such as being a potent endogenous somnogen (33), nociceptive modulator (34), anticoagulant, vasodilator, bronchoconstrictor, and an allergic and inflammatory mediator (35). L-PGDS is also a member of the lipocalin family, a group of secretory proteins transporting small hydrophobic molecules, including retinoids (36). Therefore, L-PGDS has two functions, as a PGD2-producing enzyme within cells and as a lipophilic ligand-binding protein after secretion into the extracellular space and various body fluids.
L-PGDS is located in the central nervous system, genital organs, and heart and is secreted into the cerebrospinal fluid, interphotoreceptor matrix, seminal plasma, amniotic fluid, blood stream, and urine, respectively. L-PGDS concentrations in these body fluids were changed under various pathological conditions and may be a useful clinical marker for various diseases (33, 37, 38, 39, 40). In the heart L-PGDS is expressed in cardiac myocytes and endocardial cells and secreted into the blood stream. The plasma L-PGDS concentration in coronary circulation is changed in patients with angina pectoris (38, 39). These results suggest that L-PGDS may have an important role in the heart.
Most studies have focused on the effects of PGD2 on the cardiovascular system as a measure of the function of L-PGDS. We are also interested in the function of L-PGDS as a transporter of retinoids and 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2), the metabolite of PGD2. L-PGDS can bind all-trans- and 9-cis-retinoic acid (36), which are ligands for the retinoid X receptors and the retinoic acid receptors, respectively. Interestingly, retinoid-signaling pathways are involved in cardiac development and function (41, 42, 43). Specifically, there are reports that retinoid-dependent pathways suppress the cardiac hypertrophy program (42, 43). 15d-PGJ2 is a peroxisome proliferator-activated receptor
(PPAR
) ligand and can be naturally generated from PGD2 in the presence of albumin (44). Recently, Yamamoto et al. (45) demonstrated that PPAR
activators inhibit cardiac hypertrophy by acting on cardiac myocytes. Therefore, the antihypertrophic effect of estrogen on the heart might be related to retinoid-dependent pathways, through the function of L-PGDS as a retinoid transporter and to PPAR
-dependent pathways, both through the function of L-PGDS as an enzyme generating the precursor for 15d-PGJ2 and as a transporter for 15d-PGJ2. We showed that L-PGDS mRNA expression in ovariectomized mice was reduced compared with that in sham-operated mice. These data suggest that L-PGDS expression level might relate to cardiac mass increases with age in women. Further studies are needed to test this hypothesis.
In conclusion, we have shown that estrogen regulates L-PGDS gene expression in the heart through ERß and that the L-PGDS ERE consensus sequence plays an important role in ERß selectivity. The physiological functions of L-PGDS are compatible with an important modulator of cardiac physiology and disease. Our findings also suggest that this ER selectivity provides an opportunity to study the molecular basis for receptor selectivity and has important implications in the event that this gene proves to be a potential target for therapeutic intervention.
 |
MATERIALS AND METHODS
|
---|
Animals
C57BL/6J female mice (11 wk old) were used for the short-term estrogen treatment (6 h) and the comparison between sham procedure and ovariectomy. Male double heterozygous (ER
+/-ß+/-) mice were mated with female double heterozygous (ER
+/-ß+/-) mice, resulting in ER
+/+ß+/+ = WT, ER
-/-ß+/+ = ER
- disrupted mice (ERKO), ER
+/+ß-/- = ERß-disrupted mice (BERKO), and ER
-/-ß-/- = ER
- and ß-disrupted mice (DERKO) off springs with a mixed C57BL/6J/129 background. Genotyping of the ER
and ERß has been described previously (46). These female mice (9 wk old) were used for the long-term estrogen treatment (3 wk). The mice had free access to fresh water and normal food pellets (3 wk treatment group) or soy-free food pellets (6 h treatment group and the group of comparison between sham procedure and ovariectomy). All animals, except the sham group in Fig. 4
, were ovariectomized. All studies were approved by Stockholm South Ethical Committee on animal experiments and the Ethical Committee on animal care at Gothenburg University.
Estrogen Replacement
For the acute treatment, ovariectomized mice were injected sc with 100 ìg/kg of 17ß-estradiol (Sigma, St. Louis, MO). For the chronic treatment, ovariectomized mice were injected sc with 100 µg/kg of 17ß-estradiol for 3 wk (5 d/wk). Control mice received injections of vehicle oil (olive oil or corn oil). This dose of 17ß-estradiol was referred to in Sullivans report (47). 17ß-Estradiol serum levels were measured with a RIA (Estradiol-2 kit, DiaSorin, Saluggia, Italy).
RNA Preparation
Total RNA was isolated from mouse hearts using TRIzol reagent (Invitrogen, Carlsbad, CA) and was further purified using the RNeasy Mini kit (QIAGEN, Hilden, Germany).
Affymetrix Gene Chip Analysis
For this analysis, total RNA was isolated from vehicle and estrogen-treated WT mouse hearts. Reverse transcription, second-strand cDNA synthesis, and probe generation were performed according to the Affymetrix protocol (Affymetrix, Santa Clara, CA). Murine genome U74A Genome Arrays (Affymetrix) were hybridized, washed, scanned, and analyzed according to the standard Affymetrix protocol (Affymetrix Expression Analysis Technical Manual, P/N 700218 rev.2). To identify differentially expressed transcripts, pair-wise comparison analyses were performed. Each of three estrogen treatment samples were compared with each of three vehicle samples, resulting in nine pair-wise comparisons. The results of these comparisons were indicated as increased call, decreased call, or no-change call according to Affymetrix (Microarray Suite 5.0 Expression Analysis Program). Genes in which at least seven of nine comparisons produce concordant calls (increased call or decreased call), and where the minimum average change was 1.4-fold (48), were regarded as differently expressed.
Northern Blot Analysis
Total RNA was electrophoresed in 1% agarose-2% formaldehyde gels. The gels were transferred to nylon membranes (Hybond-N+, Amersham Pharmacia Biotech, Piscataway, NJ), and the membranes were UV-irradiated. A 32P-labeled cDNA probe for a target gene was prepared by a random priming method (rediprime II, Amersham Pharmacia Biotech). Hybridization was carried out using Rapid-hyb buffer (Amersham Pharmacia Biotech). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization.
Protein Extraction and Western Blot Analysis
Mouse hearts were homogenized in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.5% sodium deoxycholate, 0.5% Nonidet P-40, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail tablet (Roche, Mannheim, Germany) per 50 ml] using a polytron homogenizer. The protein content was measured by the BCA protein assay reagent (Pierce, Rockford, IL), using BSA as a standard.
Proteins were resolved on 15% sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to polyvinylidine difluoride (PVDF) membranes (Hybond-P, Amersham Pharmacia Biotech). After transfer, the filters were incubated with a rabbit polyclonal antibody against mouse L-PGDS (0.2 µg of IgG/ml) (49). Filters were then incubated with horseradish peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech; 1:10,000 dilution). Blots were developed with enhanced chemiluminescence detection reagent (ECL, Amersham Pharmacia Biotech) following the manufacturers instructions.
Plasmid Constructs
pGL3-L-PGDS (-4037/-3265) was constructed by amplifying the mouse L-PGDS promoter region -4037/-3265, followed by subcloning into the MluI and XhoI sites of pGL3-promoter vector (Promega Corp., Madison, WI). Mutant constructs [pGL3-L-PGDS-EREm, pGL3-L-PGDS-AP-1m, and pGL3-L-PGDS-ERE (C to A)] were produced by introduction of 1- or 2-bp substitutions using the QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Synthetic single-stranded oligonucleotides corresponding to the Xenopus Vitellogenin A2 ERE and the Xenopus Vitellogenin A2 ERE (A to C), respectively, including 10 bp of flanking sequence, were annealed and ligated into the KpnI and BglII sites of the pGL3-promoter vector to produce pGL3-Xenopus Vitellogenin A2 ERE and pGL3-Xenopus Vitellogenin A2 ERE (A to C), respectively. DNA sequences of all constructs were confirmed by DNA sequencing. The primers used for site-directed mutagenesis were also used as a PGDS-EREm probe in EMSA. pSG5-mER
and pSG5-mERß, which contain murine ER
and ERß cDNAs, respectively, have been described previously (50).
Cell Culture and Transfection
293 Cells were cultured in a 1:1 mixture of Hams Nutrient mixture F12 (Invitrogen) and DMEM (Invitrogen) supplemented with 5% fetal bovine serum and 100 U penicillin/ml and 100 µg streptomycin/ml. Cells were plated in 24-well plates 24 h before transfection.
Transfection using the Superfect (QIAGEN, Chatsworth, CA) reagent was performed according to the manufacturers instructions. Briefly, 0.8 µg of pGL3-PGDS (-4037/-3265), pGL3-PGDS-EREm, pGL3-PGDS-AP-1m, pGL3-PGDS-ERE (C to A), pGL3-Xenopus Vitellogenin A2 ERE, or pGL3-Xenopus Vitellogenin A2 ERE (A to C) was cotransfected with 0.032 µg of pSG5-mER
or pSG5-mERß. A pRL-thymidine kinase control plasmid, which contains a Renilla luciferase gene, was included to control for differences in transfection efficiencies. The pSG5 vector was used to equalize plasmid concentrations. The medium was replaced with a phenol red-free mixture of F12 and DMEM containing 5% dextran-coated charcoal-treated fetal bovine serum and 100 U penicillin/ml and 100 µg streptomycin/ml upon transfection. Hormone or vehicle (in 0.1% ethanol) was added simultaneously upon transfection. Cells were harvested 24 h after transfection, and luciferase activities were determined using the Dural Luciferase Reporter Assay System (Promega Corp.) according to the manufacturers instructions.
EMSA
Recombinant human ER
or ERß, in the form of nuclear extracts from appropriately baculovirus-infected insect cells, were kindly provided by Dr. Stefan Nilsson (KaroBio AB, Sweden). Recombinant human ER
or ERß (1 µl) was incubated for 10 min at 4 C with 1 µg of poly(dI-dC) (dI-dC) in a reaction mixture containing 30 mM Tris-HCl (pH 7.4), 80 mM KCl, 0.6 mM EDTA, 0.6 mM dithiothreitol, and 12% glycerol. A double-stranded radiolabeled (1 x 105 cpm) oligonucleotide probe was added, and the reaction mixture was incubated for 30 min at room temperature. To demonstrate DNA specificity, identical reactions except for the addition of unlabeled double-stranded oligonucleotide corresponding to L-PGDS ERE/DNA-protein complexes were resolved from the free probe by electrophoresis on 5% polyacrylamide gel/0.5x Tris-borate-EDTA gels and visualized subsequent to drying and autoradiography at -70 C. Double-stranded oligonucleotides composed of the following sequences were used for EMSA:
L-PGDS ERE 5'-GCCTCTAGGTCAGTCTGCCCTCAGTTT-3';
L-PGDS EREm 5'-GGGCCTCTAGGTCAGTCTtCaCTCAGTTTCTGAG-3';
L-PGDS ERE (C to A) 5'-GCCTCTAGGTCAGTCTgaCCTCAGTTT-3';
Xenopus Vitellogenin A2 ERE 5'-AAAGTCAGGTCACAGTGACCTGATCAA-3'.
Nonspecific competitor 5'-AGCTTGCGAAAATTGTCACTTCCTGTGTACACCGA-3'.
ERE sequences are underlined. Mutated bases are shown in lowercase letters.
Statistical Analysis
All values represent mean ± SD. When significant differences are discussed, an unpaired Students t test was used.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Katarina Pettersson and Anders Ström for discussions and comments on the manuscript.
 |
FOOTNOTES
|
---|
This study was supported by grants from the Swedish Cancer Fund, KaroBio AB, a grant from the Corporated Technology Development Program of the Japan Science and Technology Corporation, and a Grant-in-Aid for Scientific Research (B) of the Ministry of Education, Culture, Sports, Science and Technology (13557016).
Abbreviations: AP-1, Activator protein 1; AP-1m, AP-1 mutant; BERKO, ERß-disrupted; 15d-PGJ2, 15-deoxy-
12,14-prostaglandin J2; DERKO, ER
- and ß-disrupted; ECM, extracellular matrix; ER, estrogen receptor; ERE, estrogen response element; EREm, ERE mutant; ERKO, ER
-disrupted; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L-PGDS, lipocalin-type prostaglandin D synthase; PPAR, peroxisomal proliferator-activated receptor; PVDF, polyvinylidine difluoride; WT, wild-type.
Received for publication January 15, 2003.
Accepted for publication June 13, 2003.
 |
REFERENCES
|
---|
- Pelzer T, Shamim A, Neyses L 1996 Estrogen effects in the heart. Mol Cell Biochem 160161:307313
- Hanes DS, Weir MR, Sowers JR 1996 Gender considerations in hypertension pathophysiology and treatment. Am J Med 101(3A):10S21S
- Schenck-Gustafsson K 1996 Risk factors for cardiovascular disease in women: assessment and management. Eur Heart J 17:28[Medline]
- Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E 1998 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 280:605613[Abstract/Free Full Text]
- Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Womens Health Initiative randomized controlled trial. JAMA 288:321333[Abstract/Free Full Text]
- Mendelsohn ME, Karas RH 1999 The protective effects of estrogen on the cardiovascular system. N Engl J Med 340:18011811[Free Full Text]
- Hayward CS, Webb CM, Collins P 2001 Effect of sex hormones on cardiac mass. Lancet 357:13541356[CrossRef][Medline]
- Lim WK, Wren B, Jepson N, Roy S, Caplan G 1999 Effect of hormone replacement therapy on left ventricular hypertrophy. Am J Cardiol 83:11321134[CrossRef][Medline]
- van Eickels M, Grohe C, Cleutjens JP, Janssen BJ, Wellens HJ, Doevendans PA 2001 17ß-Estradiol attenuates the development of pressure-overload hypertrophy. Circulation 104:14191423[Abstract/Free Full Text]
- Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-Å 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:59255930[Abstract/Free Full Text]
- Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353365[Abstract/Free Full Text]
- Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:4953[CrossRef][Medline]
- Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson J-Å 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors
and ß. Endocrinology 138:863870[Abstract/Free Full Text]
- Couse JF, Lindzey J, Grandien K, Gustafsson J-Å, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-
(ER
) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER
-knockout mouse. Endocrinology 138:46134621[Abstract/Free Full Text]
- Register TC, Adams MR 1998 Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor ß. J Steroid Biochem Mol Biol 64:187191[CrossRef][Medline]
- Lindner V, Kim SK, Karas RH, Kuiper GG, Gustafsson J-Å, Mendelsohn ME 1998 Increased expression of estrogen receptor-ß mRNA in male blood vessels after vascular injury. Circ Res 83:224229[Abstract/Free Full Text]
- Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan Jr TR, Lubahn DB, ODonnell Jr TF, Korach KS, Mendelsohn ME 1997 Estrogen inhibits the vascular injury response in estrogen receptor
-deficient mice. Nat Med 3:545548[Medline]
- Grohe C, Kahlert S, Lobbert K, Stimpel M, Karas RH, Vetter H, Neyses L 1997 Cardiac myocytes and fibroblasts contain functional estrogen receptors. FEBS Lett 416:107112[CrossRef][Medline]
- Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW, Thoren P, Smithies O, Gustafsson J-Å, Mendelsohn ME 2002 Abnormal vascular function and hypertension in mice deficient in estrogen receptor ß. Science 295:505508[Abstract/Free Full Text]
- Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM 1999 Expression profiling using cDNA microarrays. Nat Genet 21(Suppl 1):1014
- Feng X, Jiang Y, Meltzer P, Yen PM 2000 Thyroid hormone regulation of hepatic genes in vivo detected by complementary DNA microarray. Mol Endocrinol 14:947955[Abstract/Free Full Text]
- Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF 2002 Estradiol alters nitric oxide production in the mouse aorta through the
-, but not ß-, estrogen receptor. Circ Res 90:413419[Abstract/Free Full Text]
- Zhao H, Xia Z, Cai G, Du J, Zhu T, Shen L 1998 Expression of type-I collagen and matrix metalloproteinase-9 mRNA in bone of castrated adult female rats: effects of estrogen. Chin Med J (Engl) 111:551555[Medline]
- Dubey RK, Gillespie DG, Mi Z, Jackson EK 1997 Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors. Circulation 96:26562666[Abstract/Free Full Text]
- Dubey RK, Gillespie DG, Zacharia LC, Rosselli M, Imthurn B, Jackson EK 2002 Methoxyestradiols mediate the antimitogenic effects of locally applied estradiol on cardiac fibroblast growth. Hypertension 39:412417[Abstract/Free Full Text]
- Yu FX, Lin SC, Morrison-Bogorad M, Atkinson MA, Yin HL 1993 Thymosin ß 10 and thymosin ß 4 are both actin monomer sequestering proteins. J Biol Chem 268:502509[Abstract/Free Full Text]
- Slater EP, Redeuihl G, Theis K, Suske G, Beato M 1990 The uteroglobin promoter contains a noncanonical estrogen responsive element. Mol Endocrinol 4:604610[Abstract]
- Scholz A, Truss M, Beato M 1998 Hormone-induced recruitment of Sp1 mediates estrogen activation of the rabbit uteroglobin gene in endometrial epithelium. J Biol Chem 273:43604366[Abstract/Free Full Text]
- Garcia-Fernandez LF, Urade Y, Hayaishi O, Bernal J, Munoz A 1998 Identification of a thyroid hormone response element in the promoter region of the rat lipocalin-type prostaglandin D synthase (ß-trace) gene. Brain Res Mol Brain Res 55:321330[Medline]
- Garcia-Fernandez LF, Iniguez MA, Eguchi N, Fresno M, Urade Y, Munoz A 2000 Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells. J Neurochem 75:460470[CrossRef][Medline]
- Mong JA, Devidze N, Frail DE, OConnor LT, Samuel M, Choleris E, Ogawa S, Pfaff DW 2003 Estradiol differentially regulates lipocalin-type prostaglandin D synthase transcript levels in the rodent brain: evidence from high-density oligonucleotide arrays and in situ hybridization. Proc Natl Acad Sci USA 100:318323[Abstract/Free Full Text]
- Urade Y, Hayaishi O 2000 Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase. Biochim Biophys Acta 1482:259271[Medline]
- Urade Y, Hayaishi O 1999 Prostaglandin D2 and sleep regulation. Biochim Biophys Acta 1436:606615[Medline]
- Eguchi N, Minami T, Shirafuji N, Kanaoka Y, Tanaka T, Nagata A, Yoshida N, Urade Y, Ito S, Hayaishi O 1999 Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice. Proc Natl Acad Sci USA 96:726730[Abstract/Free Full Text]
- Matsuoka T, Hirata M, Tanaka H, Takahashi Y, Murata T, Kabashima K, Sugimoto Y, Kobayashi T, Ushikubi F, Aze Y, Eguchi N, Urade Y, Yoshida N, Kimura K, Mizoguchi A, Honda Y, Nagai H, Narumiya S 2000 Prostaglandin D2 as a mediator of allergic asthma. Science 287:20132017[Abstract/Free Full Text]
- Tanaka T, Urade Y, Kimura H, Eguchi N, Nishikawa A, Hayaishi O 1997 Lipocalin-type prostaglandin D synthase (ß-trace) is a newly recognized type of retinoid transporter. J Biol Chem 272:1578915795[Abstract/Free Full Text]
- Hirawa N, Uehara Y, Ikeda T, Gomi T, Hamano K, Totsuka Y, Yamakado M, Takagi M, Eguchi N, Oda H, Seiki K, Nakajima H, Urade Y 2001 Urinary prostaglandin D synthase (ß-trace) excretion increases in the early stage of diabetes mellitus. Nephron 87:321327[CrossRef][Medline]
- Eguchi Y, Eguchi N, Oda H, Seiki K, Kijima Y, Matsu-ura Y, Urade Y, Hayaishi O 1997 Expression of lipocalin-type prostaglandin D synthase (ß-trace) in human heart and its accumulation in the coronary circulation of angina patients. Proc Natl Acad Sci USA 94:1468914694[Abstract/Free Full Text]
- Urade Y, Eguchi Y, Eguchi N, Kijima Y, Matsuura Y, Oda H, Seiki K, Hayaishi O 1999 Secretion of lipocalin-type prostaglandin D synthase (ß-trace) from human heart to plasma during coronary circulation. Adv Exp Med Biol 469:4954[Medline]
- Hirawa N, Uehara Y, Yamakado M, Toya Y, Gomi T, Ikeda T, Eguchi Y, Takagi M, Oda H, Seiki K, Urade Y, Umemura S 2002 Lipocalin-type prostaglandin D synthase in essential hypertension. Hypertension 39:449454[Abstract/Free Full Text]
- Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM 1994 RXR
mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev 8:10071018[Abstract]
- Zhou MD, Sucov HM, Evans RM, Chien KR 1995 Retinoid-dependent pathways suppress myocardial cell hypertrophy. Proc Natl Acad Sci USA 92:73917395[Abstract]
- Wu J, Garami M, Cheng T, Gardner DG 1996 1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes. J Clin Invest 97:15771588[Abstract/Free Full Text]
- Fitzpatrick FA, Wynalda MA 1983 Albumin-catalyzed metabolism of prostaglandin D2. Identification of products formed in vitro. J Biol Chem 258:1171311718[Abstract/Free Full Text]
- Yamamoto K, Ohki R, Lee RT, Ikeda U, Shimada K 2001 Peroxisome proliferator-activated receptor
activators inhibit cardiac hypertrophy in cardiac myocytes. Circulation 104:16701675[Abstract/Free Full Text]
- Vidal O, Lindberg MK, Hollberg K, Baylink DJ, Andersson G, Lubahn DB, Mohan S, Gustafsson J-Å, Ohlsson C 2000 Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 97:54745479[Abstract/Free Full Text]
- Sullivan Jr TR, Karas RH, Aronovitz M, Faller GT, Ziar JP, Smith JJ, ODonnell Jr TF, Mendelsohn ME 1995 Estrogen inhibits the response-to-injury in a mouse carotid artery model. J Clin Invest 96:24822488[Medline]
- Yue H, Eastman PS, Wang BB, Minor J, Doctolero MH, Nuttall RL, Stack R, Becker JW, Montgomery JR, Vainer M, Johnston R 2001 An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression. Nucleic Acids Res 29:E41E44
- Mizoguchi A, Eguchi N, Kimura K, Kiyohara Y, Qu WM, Huang ZL, Mochizuki T, Lazarus M, Kobayashi T, Kaneko T, Narumiya S, Urade Y, Hayaishi O 2001 Dominant localization of prostaglandin D receptors on arachnoid trabecular cells in mouse basal forebrain and their involvement in the regulation of non-rapid eye movement sleep. Proc Natl Acad Sci USA 98:1167411679[Abstract/Free Full Text]
- Pettersson K, Grandien K, Kuiper GG, Gustafsson J-Å 1997 Mouse estrogen receptor ß forms estrogen response element-binding heterodimers with estrogen receptor
. Mol Endocrinol 11:14861496[Abstract/Free Full Text]