© 1997 American Heart Association, Inc.
Articles |
Effects of Estrus Cycle, Ovariectomy, and Treatment with Estrogen, Tamoxifen, and Progesterone on Apolipoprotein(a) Gene Expression in Transgenic Mice
From the Falk Cardiovascular Research Center, Stanford University School of Medicine, Palo Alto, Calif (B.R.Z., R.M.L.) and Cardiovascular Department, Berlex Biosciences, Richmond, Calif (K.K., G.M.R.).
Correspondence to Gabor M. Rubanyi, MD, PhD, Cardiovascular Department, Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804-0099.
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Abstract |
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Abstract Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.
Key Words: apolipoprotein(a) • gene regulation • estrogen • lipoprotein(a) • atherosclerosis
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Introduction |
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Lipoprotein(a) (Lp(a)) is an atherogenic lipoprotein that consists of apolipoprotein(a) (apo(a)), a large glycoprotein with significant homology to plasminogen, covalently linked to apolipoprotein B-100 (apoB-100), the sole protein component of LDL.1,2 Numerous studies have indicated that Lp(a) is an independent risk factor for cardiovascular diseases associated with atherosclerosis and its sequelae, including myocardial infarction, restenosis, peripheral vascular disease, and stroke.3–8
In contrast to other lipoproteins, interindividual plasma levels of Lp(a) vary nearly 1000-fold. Levels of Lp(a) are genetically determined, linked almost solely to the apo(a) gene locus, and are chiefly governed by biosynthetic rate rather than catabolism.9–14 Alterations in diet and treatment with most lipid-lowering agents have little effect on plasma levels of this atherogenic lipoprotein.
One of the few classes of agents that have been shown to significantly lower Lp(a) levels are sex steroid hormones. Even though the large individual variation in Lp(a) levels may mask such effects in population studies, two studies reported 8% to 13% population-based increases after menopause.15,16 Longitudinal studies following individuals with hormone replacement therapy have reported reduction in Lp(a) of as much as 50%.17–20
Analysis of the mechanism of action of endogenous and exogenous estrogens on circulating levels of apo(a) and on apo(a) gene expression have been hampered by lack of an appropriate animal model due to limited phylogenetic distribution of apo(a). A line of transgenic mice that expresses the human apo(a) transgene in liver has been created by the introduction of 270 kb of yeast artificial chromosome (YAC) containing 110 kb of an intact human apo(a) gene and more than 60 kb of both 5' and 3' sequences flanking the apo(a) gene.21 Male apo(a) YAC transgenic mice were shown to have a marked reduction in plasma levels of apo(a) on sexual maturation and to have a dramatic rise in plasma levels of apo(a) following orchidectomy. Treatment of orchidectomized mice with testosterone caused plasma levels of apo(a) to decrease to those observed prior to castration, and testosterone was demonstrated to regulate plasma concentration of apo(a) at the level of mRNA.21 In the present study, we investigated the effects of ovariectomy, estrus cycle, and treatment with ethinyl estradiol, tamoxifen, and progesterone on plasma apo(a) levels and liver apo(a) gene expression in female apo(a) YAC transgenic mice. The results show for the first time that estrogen, but not progesterone, lowers plasma apo(a) levels by suppression of apo(a) mRNA level, a mechanism that may contribute to the cardiovascular protective action of the ovarian sex steroid hormone.
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Methods |
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Animals, Estrus Cycle Determination and Experimental Design
Transgenic mice of the FVB background contained the human apo(a) gene and flanking DNA on a 270-kb YAC were produced earlier.21 All animals were housed in accordance with institutional guidelines (constant temperature; 12-hour dark/light cycle; standard mouse chow and water ad libitum). At 9 weeks of age, female mice were surgically gonadectomized to study the effect of hormonal "withdrawal" and were allowed at least 2 weeks postoperative recovery.
Estrus cycle of intact females was monitored daily. Microscopical analysis of the vaginal smears taken by small cotton swabs at the same time every day. Only animals exhibiting consistent 4-day cycles were used for measurements of plasma apo(a) levels. Estrus cycle was followed for a period of 3 full cycles prior to sampling blood by tail bleeding.
Two weeks after ovariectomy hormonal treatment of female mice was started with daily subcutaneous injections of either 5 µg of ethinyl estradiol (n=7),22 125 µg of 4-OH-tamoxifen (n=3),22 3 mg of progesterone (n=4)23, or 3 mg of progesterone and 5 µg of ethinyl estradiol together (n=4). Ovariectomized control animals were injected with vehicle alone (benzylbenzoate:castor oil/1:9). Mice were treated for a total of 14 days, then sacrificed. Blood samples were taken on the 7th and 14th day of treatment. Liver and uterus were dissected for further analyses after sacrifice of the animals on the 14th day of treatment.
Collection of Blood and Determination of Plasma Apo(a)
Levels
Blood was collected by tail bleeding during treatment and by
cardiac puncture at the end of the study into a tube containing
K+EDTA. Plasma was separated by
centrifugation of the whole blood for 10 minutes at
10 000 rpm at 21°C. Apo(a) levels were determined using the Macra
Elisa Kit (Strategic Diagnostic). All measurements were
made in duplicate.
Determination of Uterine Weight
Uterine weight was used to assess the efficacy of treatment with
exogenous estrogen and to compare drug treatment with
physiological hormonal effects. Uteri were isolated
from control (not ovariectomized) and ovariectomized vehicle or
hormone-treated mice at the end of the 2 weeks of treatment. Organs
were cleaned from adherent connective tissues and weighed. After
measuring wet organ weight, uteri were placed on aluminum foil and
dried in an oven for 24 hours. At that time dry organ weight was
determined.
RNA Isolation and Northern Analysis
Livers were frozen immediately after isolation from the animals
in liquid nitrogen and stored at -80°C. Total RNA was isolated by
Ultraspec-II RNA isolation system (Biotech Laboratory Inc.) according
to the manufacturer's protocol. For Northern blot analysis,
RNA samples were denatured at 60°C and electrophoresed through a
1.3% formaldehyde agarose gel followed by blotting to nylon membranes
and irradiation by ultraviolet light. Membranes were hybridized to
[32P]dCTP-labeled probes for either apo(a) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by
standard methods overnight at 42°C.24 The
membranes were washed once in 2 x SSC + 0.2% SDS for 30 min at
room temperature and twice with 2 x SSC + 1% SDS for 30 minutes
at 65°C. Membranes were then exposed to a phosphorimage plate
(Bio-Imaging Analyzer, BAS1000 MacBAS, Fuji Photo Film Co.,
Ltd.) for 12 hours. The phosphorimage was than scanned and
analyzed (Bio-Imaging Analyzer, BAS1000 MacBAS, Fuji
Photo Film Co., Ltd.). RNA levels were quantitated after normalization
to GAPDH. The cDNA probes used for hybridization were an
EcoRI fragment (4.0-kb) of human
apo(a)2 and a 1.0-kb fragment of human GAPDH
(Clontech Laboratories, Inc.). The cDNA probes were labeled with
[32P]dCTP using a random priming DNA labeling
kit according to the instruction of the manufacturer (Prime-a-Gene
Labeling System, Promega Corp.).
Materials
17
-Ethinyl estradiol, progesterone, and the vehicle were
obtained from Sigma. 4-OH-Tamoxifen was obtained from Aldrich.
Statistical Analysis
Values are presented as mean±SEM. Comparisons between
two groups were made by unpaired Student's t test.
Comparisons between multiple treatment groups were made by ANOVA and
Newman-Keuls tests. Differences were considered statistically
significant at P<.05.
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Results |
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Effects of Ovariectomy and Estrus Cycle
Nine female transgenic mice were followed with serial plasma apo(a) levels measured on a biweekly basis for 9 weeks prior to ovariectomy. The mean±SEM plasma apo(a) level was 20.6±1.3 mg/dL at week 5, 21.3±1.1 mg/dL at week 7, and 17.7±2.1 mg/dL at week 9. To investigate the influence of endogenous hormones on plasma apo(a) levels, effects of the estrus cycle were studied in 12 postpubertal female transgenic mice. Eleven mice were noted to be cycling synchronously over a period of three cycles. One mouse had a cycle of 5 or 6 days and was eliminated from the group. A distinct pattern was noted over the course of the 4-day cycle with the largest and statistically significant difference between day 1 (diestrus) and day 3 (estrus) (Fig 1
). This decline occurs during rising
serum estrogen levels during the estrus cycle of mice (day
1=diestrus: 21±6.5 pg/mL; day 2=proestrus: 54±8.5
pg/mL; day 3=estrus: 34±7 pg/mL; day 4=metestrus: 25±8
pg/mL).25
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Effects of Treatment With Estrogen, Progesterone, Estrogen, and
Progesterone, and Tamoxifen
Treatments of mice started on the 11th week, 2 weeks after
ovariectomy, when the mean plasma apo(a) level was 19.8±0.7
mg/dL. After 2 weeks of daily subcutaneous administration of
vehicle only, animals showed an increased plasma level of apo(a)
(pretreatment level: 21.0±4.3 mg/dL, 2 weeks of vehicle
treatment: 30.6±6.1 mg/dL, n=7, Fig 2). After 1-week treatment, the mean
plasma concentration of apo(a) in the ethinyl estradiol (5
µg/animal/day) treated mice (n=7) and the
4-OH-tamoxifen (125 µg/animal/d)-treated mice (n=3)
declined significantly (P<.05) from a pretreatment level of
22.3±2.4 mg/dL to 1.2±0.5 mg/dL and from 20.4±1
mg/dL pretreatment level to 6±1.4 mg/dL
(P<.05 versus estradiol-treated mice), respectively. At the
end of the second week of treatment, plasma apo(a) level remained
significantly suppressed in the ethinyl estradiol group (2.7±1.2
mg/dL; P<.05) (Fig 2). Plasma levels of apo(a)
decreased further in the tamoxifen-treated group to the level of
3.3±1.9 mg/dL, not different from the ethinyl estradiol
group at this time point (Fig 2). Animals (n=4) treated with
progesterone only (3 mg/animal/day) for 2 weeks, had
an increase in mean apo(a) levels from 21.3±1.8 mg/dL to
40.5±10.9 mg/dL (Fig 2), similar to vehicle treated controls.
The mice treated with the combination of estrogen and progesterone
(n=4) had a decline in the mean plasma apo(a) level during the two
weeks period from 20.2±5.0 mg/dL to 12.3±8.6 mg/dL
(P<.05) (Fig 2).
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Northern analysis of mRNA derived from the liver of transgenic
mice after two weeks treatment revealed that levels of apo(a) mRNA are
significantly reduced in the estrogen, tamoxifen, and in the estrogen
and progesterone treated animals compared to vehicle treated controls.
Levels of mRNA were unchanged in mice treated with progesterone alone.
Decrease of plasma level of apo(a) was associated with decrease in
liver apo(a) mRNA level in all groups studied (Fig 2
).
Treatment of ovariectomized transgenic mice with a 5 µg daily injection of ethinyl estradiol for two weeks restored uterine dry weight to nonovarectomized control weights. Mean uterine dry weights were 15±4.0 mg, 6±1.0 mg, and 16±2.0 mg in the control, ovariectomized, and ethinyl estradiol-treated mice, respectively.
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Discussion |
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A number of studies have attempted to define the relationship between sex steroid hormones and plasma levels of Lp(a). These studies have consistently demonstrated that treatment with exogenous estrogens decreases plasma levels of Lp(a).26–28 The mechanism underlying this effect has to date not been elucidated. Results of this study demonstrate that treatment of ovariectomized female apo(a) YAC transgenic mice with estrogen and the partial estrogen receptor agonist, tamoxifen, results in a decrease in plasma apo(a) levels due to reduction in hepatic apo(a) mRNA.
Population studies have failed to demonstrate a significant relationship between endogenous sex steroid hormones and levels of Lp(a). Given the 1000-fold variation in plasma Lp(a) concentration in humans, it is difficult to demonstrate control of Lp(a) in cross-sectional studies. Despite these limitations, the Framingham Offspring Study found an 8% increase in mean Lp(a) concentration after menopause and the ARIC Study, found a 13% increase.15,16 In premenopausal women with ovulatory menstrual cycles plasma apo(a) levels increased significantly in four out of 15 patients after the LH surge and throughout the luteal phase associated with lower estradiol plasma levels,29 similar to the inverse relationship found between plasma levels of 17ß-estradiol and apo(a) during the estrus cycle of mice in the present study.
Several cross-sectional and longitudinal studies have demonstrated that administration of naturally occurring and synthetic sex steroid hormones have a significant and consistent effect in lowering plasma concentrations of Lp(a) in humans.30,31 Research on various regimens of postmenopausal hormone replacement, including estrogen only, estrogen and progesterone, norethisterone, and tibuone, has shown a 14% to 50% reduction in plasma levels of Lp(a).17–20 Administration of estrogens to males with prostatic cancer resulted in a 50% decrease in plasma Lp(a) levels.27 Tamoxifen, an agent that is both an estrogen receptor agonist and antagonist has also been shown to decrease Lp(a) by 40% in healthy postmenopausal women.28
The apo(a) YAC transgenic mouse has proved to be a useful model to study the effects of sex steroid hormones on regulation of apo(a) levels. A previous report has described the marked differences noted between male and female YAC transgenic mice, an observation which itself points to a potentially important role of sex steroids in the regulation of apo(a) levels.21
Unlike the effects of gonadectomy in the male apo(a) YAC transgenic mice,21 ovariectomy did not change mean plasma levels in the females, but rather caused a more subtle change that consisted of diminishing the variation around the mean apo(a) plasma level. This led us to hypothesize that we had eliminated the effects of estrus cycle on levels of apo(a). We were indeed able to demonstrate a characteristic pattern of changes throughout the estrus cycle: plasma levels of apo(a) decreased significantly between diestrus and estrus when plasma concentrations of 17ß-estradiol were reported to increase in mice.25 These data suggest that changes in endogenous levels of female sexual steroid hormones (especially 17ß-estradiol) may play an important role in the regulation of apo(a) levels. In contrast to the plasma apo(a) lowering effect of elevated plasma 17ß-estradiol during estrus, lowering plasma 17ß-estradiol by ovariectomy had no significant effect on mean plasma apo(a) levels. This observation suggests that cessation of ovarian function may cause changes in other factors which also regulate apo(a) gene expression and may counterbalance the apo(a) elevating effect of reduced circulating estrogen levels.
Treatment of ovariectomized female mice for 2 weeks with replacement doses of ethinyl estradiol (as assessed by the restoration of uterine weight in ovariectomized animals to preovariectomy level) or with tamoxifen, a partial estrogen receptor agonist, resulted in marked decreases in plasma levels of apo(a) that are seen as early as 1 week and continued to be significantly decreased at 2 weeks. Although both tamoxifen and estrogen caused a significant suppression of plasma apo(a) level after the first week of the study, treatment with estradiol was more effective than tamoxifen. This difference disappeared by the end of the 2-week treatment period, but our data suggest the possibility of additional effect of tamoxifen beyond transcription.
The mechanism underlying estrogen-induced lowering of plasma apo(a)
levels appears to be at the level of hepatic apo(a) mRNA, because it
showed similar changes. Although the exact mechanism of suppressing
apo(a) gene expression is not known, the estrogen receptor may play a
crucial role. Indeed in a human hepatoma cell line (HepG2), transfected
with 1.2 kb of the 5'-flanking sequence of the apo(a) gene, but lacking
the estrogen receptor, 17ß-estradiol had no effect on apo(a) gene
expression (our unpublished observation). After activation by its
ligands (eg, estrogen and tamoxifen) estrogen receptor may suppress
gene expression via direct binding to the genomic DNA or indirectly via
interaction with other proteins (eg, transcription factors). In vitro
studies have demonstrated that inhibition of the IL-6 gene expression
in HeLa and murine bone marrow cells by estrogen is mediated through
the estrogen receptor in the absence of high affinity DNA
binding.32–33 A recent study has demonstrated
that this inhibition is mediated by a protein-protein interaction
between the estrogen receptor and the transcription factors NF-
B and
C/EBPß.34 In this regard, our findings are
consistent with the dual activity of tamoxifen as estrogen
receptor antagonist and agonist. A recent study using rat
GC3 cells showed that, when estrogen causes gene activation, tamoxifen
acts as an antagonist by binding to the estrogen receptor
and reducing its interaction with its high affinity DNA sites. In
contrast, when estrogen causes repression (as here with apo(a)),
tamoxifen can act as an agonist through a pathway involving
protein-protein interaction with other transcription
factors.35
The present study also showed that the other ovarian sex hormone, progesterone, had no effect on apo(a) gene expression or plasma levels of apo(a) in transgenic mice. Progesterone also did not affect the apo(a)-lowering action of estrogen, when the two were given in combination. These findings suggest that the progestin component of combined hormone replacement therapy preparations may not influence the beneficial effect of estrogen on plasma Lp(a) levels in postmenopausal women.
In summary, the present study shows, for the first time, that estrogen and tamoxifen, but not progesterone, suppress expression of the human apo(a) gene and consequently lower circulating plasma levels of apo(a) in transgenic mice. This effect of estrogen may contribute to its antiatherosclerotic and cardiovascular protective action observed in postmenopausal women.36,37
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Cardiovascular Department of Berlex Biosciences and National Institutes of Health Grants to R. M. Lawn and B. R. Zysow. The authors are grateful to Jeff Davis and Timothy Kenrick (Berlex Biosciences) for their dedicated work in animal husbandry and active contribution to the estrus cycle determination and daily drug treatment.
Received August 8, 1996; accepted January 1, 1997.
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F. Nassir, D. K. Bonen, and N. O. Davidson Apolipoprotein(a) Synthesis and Secretion from Hepatoma Cells Is Coupled to Triglyceride Synthesis and Secretion J. Biol. Chem., July 10, 1998; 273(28): 17793 - 17800. [Abstract] [Full Text] [PDF]
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R. Ramharack, D. Barkalow, and M. A. Spahr Dominant Negative Effect of TGF-ß1 and TNF-{alpha} on Basal and IL-6–Induced Lipoprotein(a) and Apolipoprotein(a) mRNA Expression in Primary Monkey Hepatocyte Cultures Arterioscler Thromb Vasc Biol, June 1, 1998; 18(6): 984 - 990. [Abstract] [Full Text] [PDF]
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