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Endocrinology Vol. 149, No. 2 711-716
Copyright © 2008 by The Endocrine Society

Estrogen-Enhanced Gene Expression of Lipoprotein Lipase in Heart Is Antagonized by Progesterone

Dianxin Liu, Anne Deschamps, Kenneth S. Korach and Elizabeth Murphy

Laboratories of Signal Transduction (D.L., A.D., E.M.), and Reproductive and Developmental Toxicology (K.S.K.), National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709; and Vascular Medicine Branch (A.D., E.M.), National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Elizabeth Murphy, National Institutes of Health Vascular Medicine Branch, Room 7N112, 10 Center Drive, Bethesda, Maryland 20892. E-mail: murphy1{at}niehs.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although estrogen has effects on the heart, little is known regarding which genes in the heart are directly responsive to estrogen. We have shown previously that lipoprotein lipase (LPL) expression was increased in female hearts compared with male hearts. To test whether LPL gene expression in heart is regulated by estrogen, we perfused mouse hearts from ovariectomized females with 100 nM 17β-estradiol or vehicle for 2 h, after which hearts were frozen, and RNA was isolated. The SYBR green real-time PCR method was used to detect LPL gene expression. We found that addition of 17β-estradiol to hearts from ovariectomized females resulted in a significant increase in LPL mRNA. This estrogen effect on LPL gene expression in mouse heart can be blocked by the estrogen receptor (ER) antagonist ICI 182,780 or by progesterone. We also identified a potential estrogen receptor element (ERE) enhancer sequence located in the first intron of the mouse LPL gene. The potential ERE sequence was linked to a TATA-luciferase (LUC) reporter plasmid in HeLa cells. Both ER{alpha} and ERβ stimulated strong activity on the heterologous promoter reporter in Hela cells upon estrogen addition. Both ER{alpha} and ERβ activities on the LPL ERE reporter were abrogated by the ER antagonist ICI 182,780. Progesterone also dose dependently inhibited the estrogen-mediated increase in LPL ERE reporter activity. These results show that heart LPL is an estrogen-responsive gene exhibiting an intronic regulatory sequence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANIMAL AND EPIDEMIOLOGICAL studies have suggested that estrogen is cardioprotective (1, 2, 3, 4, 5, 6, 7, 8, 9). However, a large clinical trial did not find hormone replacement therapy to reduce cardiovascular disease in postmenopausal women (10). Furthermore, many of the beneficial cardiovascular effects of estrogen have been attributed to effects on lipids or effects on the vasculature. There are few studies examining the direct effects of estrogen on the heart. In fact, direct effects of estrogen on the heart have been questioned (11).

Two distinct estrogen receptors (ERs), ER{alpha} and ERβ, mediate estrogen responses. They belong to the steroid hormone receptor superfamily, which acts as ligand activated transcription factors on downstream target genes. Both ER{alpha} and ERβ activate gene expressions by binding to promoter regions containing a consensus estrogen response element AGGTCANNNTGACCT, typically spaced by three nucleotides or nonconsensus imperfect sequences. ERs can also interact with other transcription factors, such as specificity protein 1 and activator protein 1 to regulate gene expression, through a protein-protein tethering mechanism that has been shown in limited studies to be active in vivo (12). ER{alpha} and ERβ have a similar affinity for 17β-estradiol (E2), and are reported to be expressed in human and rat cardiac myocytes and fibroblasts (13, 14, 15, 16).

A previous study in this laboratory showed that cardiac lipoprotein lipase (LPL) expression is lower in females from ERβ knockout mice compared with wild-type females or females from ER{alpha} knockout mice (17). This suggests that estrogen may play a role in LPL gene expression in the heart. LPL controls fatty acid uptake through hydrolysis of triglyceride in chylomicrons and very large density lipoproteins. This enzyme is ubiquitously expressed with higher expression levels in heart, liver, and adipose tissue. Cardiac muscle has the greatest expression of LPL on a per gram basis (18). The heart requires a large amount of energy for contraction and relaxation, and under normal physiological conditions, fatty acids are a preferred substrate. Thus, LPL plays a crucial role in releasing free fatty acids from very large density lipoproteins and chylomicrons. Mice with global knockout of LPL exhibit extremely high blood triglyceride concentration and died within 48 h of birth. Overexpression of LPL in a single tissue such as heart, skeletal muscle, or adipose tissue can rescue these global LPL knockout mice from death. Mice expressing LPL only in the heart are able to maintain normal lipid levels (19). Cardiac-specific loss of LPL causes the heart to switch substrate preference from fatty acids to mainly glucose, a reversion to a fetal phenotype. Cardiac-specific LPL-knockout hearts exhibit decreased triglyceride and free fatty acid content (20). Together, these data suggest that LPL plays an important role in heart energy metabolism.

In this study we tested whether estrogen addition directly to heart can alter the expression of cardiac genes. We perfused isolated hearts from ovariectomized (OVX) females with estrogen for 2 h to determine if there are any direct effects of ER-mediated estrogen action on gene expression in the heart. We found that estrogen stimulates LPL expression in the OVX mouse heart. We further report that progesterone addition blocks the estrogen-dependent stimulation of LPL. Moreover, we identified that there are functional estrogen responsive elements located in the LPL intron 1 indicating a novel intronic gene regulation of LPL by ER action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health. The OVX C57B6 mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA); these mice were OVX at 8–9 wk, and perfusion experiments were conducted 3 wk later.

Heart perfusion
Mice were 11–12 wk of age at the time of experiment. Mice were anesthetized with pentobarbital, then heparinized. Hearts were isolated and perfused in the Langendorff mode as reported elsewhere (8). The hearts were excised quickly and placed in ice-cold perfusion buffer. The aorta was cannulated for retrograde perfusion at a constant pressure of 90 cm of H2O. The perfusate was modified Krebs-Henseleit buffer containing (in mM) 120 NaCl, 25 NaHCO3, 5.9 KCl, 1.2 MgSO4, 1.75 CaCl, and 10 glucose, gassed with 95% O2 and 5% CO2 at 37 C. The hearts were perfused with vehicle or 100 nM E2 in the Krebs buffer for 2 h. A final concentration of 500 nM ICI 182,780 (Tocris, Ellisville, MO) or 20 µM progesterone (Sigma-Aldrich, St. Louis, MO) dissolved in ethanol was added as indicated in the perfusion experiments. We chose 2 h because the perfused heart preparation is only stable for approximately 4 h. At the end of the treatment, hearts were snap frozen in liquid nitrogen.

RNA extraction and real-time PCR
Total RNA was extracted using the QIAGEN RNeasy Midi Kit (QIAGEN, Inc., Valencia, CA). The level of gene expression was determined by real-time PCR. The primer sequences used in RT-PCR are shown in Table 1Go. First-strand cDNA synthesis was conducted for 60 min at 48 C in a 10-µl reaction system containing 50 ng total RNA, 5.5 mM MgCl2, 2 mM dNTPs, 2.5 µM random hexamers, 4 U RNase inhibitor, and 12.5 U reverse transcriptase. The PCR was performed in a total volume of 50 µl, including the 10 µl RT reaction, to which was added 4 mM MgCl2, 8 mM dNTPs, 0.4 µM gene specific primers, and 2.5 U AmpliTaq Gold DNA polymerase (PE Biosystems, Applied Biosystems, Foster City, CA). The reaction was performed and analyzed by Applied Biosystems PRISM 7700 detection system. The level of specific mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). OVX did not affect GAPDH levels. Each reaction was performed in duplicate.


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  		TABLE 1. Primer sequence

 
Reporter construct and activity assay
The LPL gene intron 1 contains two nontypical estrogen responsive elements. Two primers were designed as follows: LPL+604 F 5'-CGGGTACCAAGCTTGTGGAGACGCAGAGGCCGAGCAAA-3' and LPL-ERE2R 5'-CGCTCGAGAGGCGTCTGGACCCGCAGTCCCA-3'. The plasmid pBluescript containing LPL promoter (–1980 to +1800) was kindly provided by Dr. Jeffrey Gimble (Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA). The fragment containing LPL estrogen responsive elements was obtained by PCR using the pBluescript as template and cloned into Stratagene’s commercial reporter plasmid pLuc-MCS (Stratagene, La Jolla, CA). Hela cells were cultured in E-MEM medium with 10% charcoal-stripped serum. Twenty-five nanograms of ER{alpha} or β expressing vector and internal control Renilla luciferase vector p-RL-TK (Promega Corp., Madison, WI) together with 300 ng LPL reporter were cotransfected in six-well plates according to the procedure of QIAGEN Effectene transfection method. E2 (10 nM) was added for 24 h in the cell culture medium to activate LPL reporter. When indicated, an ER antagonist ICI 182,780 (Tocris) was added to block ER{alpha} or β activity. The positive control plasmids 1x ERE-TATA-Luc and 3x EREs-TATA-Luc were provided by Donald McDonnell (Department of Pharmacology and Cancer Biology, Duke University, Durham, NC). The Dual-Luciferase Reporter Assay System was purchased from Promega.

Statistics
Data are shown as mean ± SE. Means were compared by one-way ANOVA. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heart perfusion
We were interested in evaluating whether addition of estrogen had direct effects on gene expression in the heart. We examined expression of ERs in a saline-perfused mouse heart using real-time PCR. The primer sequences are shown in Table 1Go. Expression was normalized to GAPDH levels. We found that the mouse heart expresses ER{alpha} and β; however, the ERβ transcript levels are much lower than ER{alpha} (Fig. 1Go).


Figure 1
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  		FIG. 1. ER{alpha} and β transcripts in mouse heart. A, ER{alpha} expression level. B, ERβ expression level. The control is vehicle perfused ovary intact mice. ovx, OVX mice treated with vehicle. ovxE2, OVX mice treated with 100 nM E2. All the hearts (n = 4 per group) were perfused with Krebs buffer with or without E2 for 2 h. ER{alpha} and ERβ expression was normalized to GAPDH. There is no significant difference in either ER{alpha} or ERβ expression among these three groups.

 
To address whether estrogen regulates gene expression in the heart, we directly perfused isolated hearts from OVX females with 100 nM E2 for 2 h, after which time we extracted RNA for real-time PCR measurements. To confirm that the OVX mice indeed have low estrogen levels, we measured uterine weight. As shown in Table 2Go, OVX significantly decreased uterine weight. Interestingly, OVX also increased body weight. Two hours of estrogen perfusion had no effect on either ER{alpha} or ERβ mRNA levels (Fig. 1Go).


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  		TABLE 2. The body and uterine weight after OVX

 
Because we had previously found a male-female difference in expression of LPL, we examined whether estrogen perfusion of the heart would result in increased expression of LPL mRNA. As shown in Fig. 2Go, perfusion of an isolated heart from an OVX female for 2 h with a buffer containing 100 nM E2 resulted in an increase in expression of LPL compared with OVX hearts perfused with vehicle. Estrogen addition to a heart from an ovary intact animal does not alter LPL expression (data not shown). These data suggest that the estrogen activation of LPL is not lost during the short time period of the heart perfusion. To test if this estrogen-induced increase in cardiac LPL mRNA is mediated by estrogen binding to the ER, the ER antagonist ICI 182,780 was added in the perfusate with estrogen. As shown in Fig. 3Go, addition of ICI 182,780 blocks the estrogen dependent stimulation of LPL gene expression. These data are consistent with the estrogen regulation of LPL. However, surprisingly, when we compared hearts from ovary intact females perfused with vehicle to OVX females perfused with vehicle, we found no difference in expression of LPL (Fig. 2Go). Because ovaries produce progesterone as well as estrogen, we examine the effects of progesterone on the estrogen-mediated increase in LPL expression. As shown in Fig. 4Go, addition of 20 µM progesterone to the perfusion buffer containing estrogen inhibited the increase in LPL mRNA observed with estrogen alone in OVX hearts. Furthermore, perfusion of hearts from OVX female with progesterone perfusion alone for 2 h significantly reduced the level of LPL mRNA compared with hearts from OVX females perfused with vehicle (Fig. 4Go).


Figure 2
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  		FIG. 2. Estrogen stimulates heart LPL gene expression in an OVX mouse. The control sample is the vehicle perfused ovary intact heart. The hearts were perfused with Krebs buffer with vehicle or E2 for 2 h. The hearts from OVX mice had similar LPL expression level compared with control. E2 increases LPL expression in hearts from OVX mice. *, Significantly different compared with OVX. n = 4 for each group. ovx is OVX mouse hearts treated with vehicle. ovxE2 is OVX mouse hearts treated with 100 nM E2.

 

Figure 3
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  		FIG. 3. ICI 182,780 inhibits LPL gene expression. ICI 182,780 is an ER antagonist. The hearts were perfused with Krebs buffer with 100 nM E2 alone (ovxE2) or 100 nM E2 combined with 500 nM ICI (ovxE2ICI) or 500 nM ICI alone (ovxICI). ICI blocks E2 activity on heart LPL gene expression. ICI alone inhibits mouse heart LPL gene expression compared with ovx group. *, Significantly different than ovx. #, Significantly different than ovxE2. n = 4 for each group.

 

Figure 4
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  		FIG. 4. Progesterone inhibits LPL expression in hearts from OVX mice. Hearts from OVX mice were perfused with vehicle (ovx), with 100 nM E2 (ovxE2) with 100 nM E2 and 20 µM progesterone (ovxE2P), or with 20 µM progesterone (ovxP). *, Significantly different than ovx. #, Significantly different than ovxE2. n = 4 for each group.

 
Reporter assay studies
We next examined potential mechanisms by which estrogen might stimulate expression of LPL. A typical estrogen regulatory response element is two inverted half-sites of GGTCA spaced by three nucleotides. In the mouse LPL gene intron 1, there exists two nontypical estrogen receptor elements (EREs). One estrogen response element overlaps with an identified functional LXRE sequence with two direct repeats of GGTCA containing four nucleotides spacers (Fig. 5AGo). To test if the EREs are functional or not, we linked the fragment to a commercial TATA-LUC reporter. After cotransfected with an ER{alpha} expression vector, no activity was found on the empty reporter without or with estrogen. However, there was a moderate increase in LPL heterologous promoter activity even without estrogen. With addition of estrogen, ER{alpha} revealed a dramatically increased activity on the LPL reporter (Fig. 5BGo). When the ERβ expression vector was cotransfected with the LPL reporter, upon the addition of estrogen, ERβ showed significant activity on the reporter (Fig. 5CGo). Both ER{alpha} and ERβ activities on the LPL reporter with estrogen were blocked by the ER-specific antagonist ICI 182,780 (Fig. 6Go, A and B).


Figure 5
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  		FIG. 5. ERs transactivate LPL reporter upon the presence of E2. A, Sequence of estrogen responsive elements in LPL gene intron1. B, ER{alpha} activates LPL ERE-reporter activity in Hela cell transfection system. With or without E2, ER{alpha} has no activity on empty TATA reporter. Without E2, ER{alpha} has moderate activity on the promoter reporter because of its N-terminal ligand-independent transactivity. With the ligand E2 at 10 nM, ER{alpha} has strong activity on the LPL ERE reporter. C, ERβ activates LPL ERE-reporter activity in the Hela cell transfection system. Without E2, ERβ has no activity on the reporter activity. With the ligand E2 at 10 nM, ERβ has strong activity on the LPL ERE reporter. *, Statistically different than in the absence of E2 and the absence of ER. n = 6 for each condition.

 

Figure 6
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  		FIG. 6. ICI 182,780 blocks ER activity on LPL ERE-TATA-LUC transactivity. A, ICI abrogates ER{alpha} activity on LPL reporter. With 10 nM E2, ER{alpha} has strong activity on the LPL reporter. After 100 nM ICI was added in the E2 treated transfection system, the ER{alpha} activity on the LPL reporter was totally blocked. B, ICI abrogates ERβ activity on LPL reporter. With 10 nM E2, ERβ also has strong activity on the LPL reporter. After ICI was added as 100 nM in the E2 treated transfection system, the ERβ activity on the LPL reporter was blocked. *, Significantly different than control. n = 6 for each group.

 
We then performed experiments to examine the dose response of estrogen on the LPL reporter. The traditional 1x ERE- and 3x ERE-TATA-LUC were used as controls. Estrogen produced a dose-dependent increase of the ER{alpha} mediated activity on the 3x ERE reporter. The LPL reporter and 1x ERE show a similar estrogen-dose response (Fig. 7AGo). With the ERβ transfection system, estrogen showed low activity with the 1x ERE. The LPL reporter and 3x ERE also have similar response increases to the estrogen dose (Fig. 7BGo). These data suggest that ERβ has the potential to regulate the LPL estrogen response element.


Figure 7
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  		FIG. 7. E2 dose response effect on ER mediated reporter activity in Hela cells. The reporters of 1x ERE-TATA-LUC, 3x EREs-TATA-LUC and LPL-TATA-LUC are used in this experiment. The concentrations of E2 are as follow: 0, 0.01, 0.1, 1, 10, and 100 nM. A, E2 dose response effect on ER{alpha}-mediated reporter activity in Hela cells. In addition to estrogen responsive reporters and p-TK-Renilla as an internal control reporter, 25 ng ER{alpha} expression vector is cotransfected in the transfection system. B, E2 dose response effect on ERβ-mediated reporter activity in Hela cells. In addition to the estrogen responsive reporters and p-TK-Renilla, a 25-ng ERβ expression vector was cotransfected in the transfection system. *, Significantly different compared with zero estrogen. n = 6 for each group.

 
Because progesterone was found in Fig. 4Go to inhibit the estrogen-mediated increase in LPL mRNA, we examined whether progesterone would inhibit the effect of estrogen on LPL reporter activity. Hela cells express the progesterone receptor (21). As shown in Fig. 8AGo, when the ER{alpha} and progesterone receptor were cotransfected with the LPL reporter, addition of progesterone dose dependently inhibited the estrogen-mediated increase in LPL reporter activity. Similarly, when the ERβ and the progesterone receptors were cotransfected with the LPL reporter, addition of progesterone dose dependently inhibited the estrogen-mediated increase in LPL reporter activity.


Figure 8
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  		FIG. 8. Progesterone blocks ER activity on the LPL reporter. A, Progesterone reduces ER{alpha} activity on the LPL reporter. With 10 nM E2, ER{alpha} has a strong activity on the LPL reporter, which was set to 100%. Progesterone added with 10 nM estrogen dose dependently inhibited the estrogen-dependent increase in LPL reporter activity. B, Progesterone also reduces ERβ activity on the LPL reporter. With 10 nM E2, ERβ has a strong activity on the LPL reporter, which was set to 100%. Progesterone dose dependently inhibited the estrogen-mediated increase in the LPL reporter activity. *, Statistically different than no progesterone. n = 6 for each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although estrogen has clear effects on cardiac physiology and pathophysiology, it has been suggested that these effects are indirect and not mediated by the direct effects of estrogen on the heart. Several studies have found ER mRNA and protein in heart and myocytes (13, 14, 15), however, the presence of ERs in the heart has been questioned (11). There is also a question regarding which ER isoforms are dominant in the heart. To evaluate whether estrogen has direct effects on gene regulation in the heart, we perfused hearts from OVX females with estrogen or vehicle for 2 h and measured changes in expression of LPL. Consistent with a role for estrogen in regulating LPL in heart, we found that perfusion of hearts for 2 h with estrogen results in a significant increase in LPL mRNA in heart, as measured by real-time PCR. This estrogen-mediated increase in LPL mRNA expression appears to be dependent on the ER as it is blocked by co-perfusion with the ER antagonist ICI 182,780. ICI 182,780 alone also inhibits LPL activation. The precise mechanism is unknown, but it likely involves ERs and the recruitment of corepressors to the promoter region; thus, ICI 182,780 may be inhibiting a basal activity that is being stimulated by ligand-independent ER activation.

Somewhat surprisingly, we did not see a difference in LPL mRNA levels between ovary intact and OVX female hearts perfused with estrogen. This led us to investigate whether another ovarian hormone, such as progesterone, might oppose the estrogen-mediated increase in LPL mRNA expression. We made the novel observation that progesterone decreases LPL expression and that addition of progesterone with estrogen blocks the estrogen-mediated stimulation of LPL mRNA.

An examination of the LPL gene suggested a potential estrogen response element in intron 1 of the LPL gene. Transient transfection of potential ERE linked to a TATA-LUC reporter in Hela cells showed that both ER{alpha} and ERβ have strong activity on this ERE sequence. Moreover, we showed that this heterologous reporter activity is estrogen-concentration dependent, as is the positive control multiple 3 x or 1 x consensus ERE reporter. The increase in LPL heterologous promoter activity even without estrogen is likely due to ER{alpha} ligand-independent basal transactivity. This estrogen effect on the LPL reporter is consistent with the estrogen-mediated increase in LPL we observed in perfused heart. We further show that progesterone dose dependently inhibits the estrogen-mediated activation of the LPL reporter.

LPL is an important regulator of fatty acid uptake in the heart. Cardiac-specific loss of LPL resulting in a reduction in lipoprotein uptake into the heart results in increased glucose metabolism and cardiac dysfunction, as indicated by a decrease in fractional shortening (22). However, overexpression of LPL on the surface of the cardiomyocyte increases lipid uptake and also causes a cardiomyopathy (23). Thus, altered expression of LPL could have effects on cardiac substrate selection and cardiac function. Estrogen also has been reported to regulate LPL expression in other tissues. In human adipocytes estrogen had a biphasic response on LPL levels; relative to untreated control, LPL was increased at low levels and decreased at high estrogen levels (24). Plasma LPL levels were also reported to be elevated in females (25, 26).

In summary, direct, acute perfusion of mouse heart with estrogen results in increased expression of LPL. These data strongly support the hypothesis that estrogen has a direct effect on gene expression in the heart. We focused on estrogen regulation of LPL because we had previously shown male-female differences in LPL expression in heart and because LPL is important in cardiovascular metabolism. We report that estrogen enhances LPL by a mechanism involving the ER. We further show that progesterone opposes the estrogen-mediated increase in LPL mRNA. These data provide new insight into the complex interaction between estrogen and progesterone in regulating gene expression.


   Footnotes
 
This work was supported by the National Institutes of Health intramural program.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 1, 2007

Abbreviations: E2, 17β-Estradiol; ER, estrogen receptor; ERE, estrogen receptor element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPL, lipoprotein lipase; Luc, luciferase fluorescence; OVX, ovariectomized.

Received May 10, 2007.

Accepted for publication October 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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H. Wang and R. H. Eckel
Lipoprotein lipase: from gene to obesity
Am J Physiol Endocrinol Metab, August 1, 2009; 297(2): E271 - E288.
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