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Lipid Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • phytoestrogens • genistein • daidzein • apolipoprotein A-I • gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kannel et al. 1986
).
This inverse association is due to the important role played by HDL and
apo A-I in the reverse cholesterol pathway, in which HDL functions
as an acceptor of cholesterol from peripheral cells and then transports
it back to the liver where it is excreted (Havel and Kane 1989
). The incidence of CHD in Asian populations is lower than
that in Western populations, and it has been speculated that both
genetic and environmental differences may be the cause of lower CHD
risk in Asian countries (Beaglehole 1990
). Among the
environmental differences, it has been suggested that the much higher
consumption of soybean products in Asian countries may be partly
responsible for the increased protection from CHD (Tham et al. 1998
). Several studies have been conducted in both animals and
humans to test the effect of soybean on risk factors for heart disease.
Studies using monkeys as animal models of lipoprotein metabolism and
CHD have shown that diets enriched in soybean phytoestrogens
significantly increase plasma HDL cholesterol and apo A-I levels,
and decrease plasma LDL cholesterol levels (Anthony et al. 1996
). A reduction in the extent of atherosclerosis in monkeys
fed the high phytoestrogen diet, compared with the regular diet, was
also observed (Anthony et al. 1997
). In rats and
hamsters, a high soybean phytoestrogen diet effectively reduced LDL
cholesterol levels and tended to increase (P = 0.1) HDL
cholesterol levels (Balmir et al. 1996
). In humans, a
meta-analysis of the effect of soy on plasma lipid levels indicated
a clear effect on LDL cholesterol levels, with a mean reduction of
12.9%, and a modest effect on HDL cholesterol levels, with a mean
increase of 2.4% (Anderson et al. 1995
). However, some
recent studies in human subjects have shown little or no effect of
phytoestrogens on plasma lipids (Hodgson et al. 1998
,
Nestel at al. 1997
).
We showed previously that estrogen increases apo A-I gene
expression in the human hepatoma cell line Hep G2 (Lamon-Fava et al. 1999
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein, daidzein, 17-ß-estradiol (E2) and progesterone were obtained from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). All other cell culture supplies, as well as Lipofectamine and Triazol, were purchased from Life Technologies (Grand Island, NY). The pGL2 plasmid and the kit assays for the measurement of luciferase and ß-galactosidase activity were obtained from Promega (Madison, WI). 32P-UTP was obtained from NEN Life Science Products (Boston, MA).
Cell culture.
The human hepatoma cell line Hep G2 was used in all experiments. Hep G2 cells were grown in high glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS, 1% nonessential amino acids, 1% Glutamax, 100,000 U/L penicillin and 100 mg/L streptomycin in 5% CO2 atmosphere at 37°C.
For experiments, on d 1, cells were seeded in 35-mm dishes at a density
of 3.5 x 105 cells/dish using phenol red–free
DMEM/F12 media containing 10% charcoal/dextran-treated FBS,
antibiotics and media supplements as described above. On d 2, cell
monolayers were washed and incubated in serum-free media. On d 3,
either vehicle alone (ethanol or dimethyl sulfoxide) or genistein,
daidzein, E2 and progesterone were added to the
serum-free media at the concentrations described. Media for apo
A-I and protein determination were collected on d 4. Apo A-I
was measured using an ELISA as described previously (Lamon-Fava et al. 1999
).
Nuclear run-on.
Hep G2 cells were incubated for 24 h with vehicle alone (control) or 10 µmol/L genistein as described above. A total of 70 x 106 cells were used in each experiment. Nuclei were isolated by homogenization with 10 strokes in a Dounce homogenizer in 2 mL of lysis buffer [10 mmol/L Tris (pH 7.4), 3 mmol/L CaCl2, 2 mmol/L MgCl2, 8 mmol/L NP-40], followed by centrifugation at 600 g for 10 min at 4°C. Nuclei pellets were immediately frozen in liquid nitrogen in 200 µL of freezing buffer [3.8 mol/L glycerol, 50 mmol/L Tris (pH 8.3), 5 mmol/L MgCl2, 0.1 mmol/L EDTA) until assay. The in vitro labeling of nascent RNA was performed in 5 mmol/L Tris (pH 8.0), 2.5 mmol/L MgCl2, 150 mmol/L KCl, 0.25 mmol/L ATP, CTP, GTP and 9.3 MBq 32P-UTP at 30°C for 30 min. RNA was isolated with 2 mL of Triazol reagent, and then hybridized to nylon filters containing 5 µg of plasmid cDNA for ß-actin and apo A-I and 5 µg of empty plasmid pGEM-4Z.
Plasmids.
The -41AI.Luc and -256AI.Luc plasmids, which contain the -41 to +396
and the -256 to +396 region of the apo A-I gene,
respectively, cloned in front of the luciferase gene in the pGL2-basic
vector, were described previously (Lamon-Fava et al. 1999
). The RSV-ß-galactosidase vector was also described
previously (Lamon-Fava et al. 1992
).
Transfection experiments.
Transfection experiments were carried out as described previously
(Lamon-Fava et al. 1999
). Briefly, on d 1, cells were
seeded in 35-mm dishes as described above. On d 2, cell monolayers were
washed and then transfected with 0.75 µg of the
RSV-ß-galactosidase plasmid and 1.1 µg of the
-41AI.Luc plasmid or molar equivalent of the -256AI.Luc plasmid in
the presence of 8 µL of Lipofectamine reagent and
under serum-free and antibiotic-free conditions. On d 3, cells
were washed and incubated in serum-free medium containing genistein
(10 µmol/L) or vehicle (ethanol). On d 4, cells were
collected and cell extracts were stored at -70°C until
ß-galactosidase and luciferase activities were assayed.
Statistical analyses.
All statistical analyses were performed using SPSS software (version 9.0; SPSS, Chicago, IL). Experiments were conducted at least two times in duplicate. Data are presented as means ± SD. For the dose-response study, groups were compared using one-way ANOVA after values were log-transformed to adjust for differences in variation between groups. Post-hoc tests were performed using Tukey’s test. The effect of genistein on plasmid expression was tested with two-way ANOVA, with the interaction between treatment and plasmid in the model. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A dose-response effect of genistein, daidzein and
E2 on apo A-I secretion was observed, with
genistein and E2 increasing apo A-I by
approximately fivefold at the 10 µmol/L concentration
(P < 0.001). The effect of daidzein on apo A-I
secretion was lower but, at the highest concentration (10
µmol/L), daidzein still significantly increased apo
A-I concentration above control levels (P < 0.001). Progesterone did not have any effect on apo A-I secretion,
suggesting a specific estrogenic effect of E2,
genistein and daidzein.
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, Lamon-Fava et al. 1999
). To assess whether the effect of genistein on apo
A-I is similar to that of estrogen, a nuclear run-on experiment
was performed. As shown in Figure 2
, compared with the control, genistein activated the transcription of
the apo A-I gene relative to the housekeeping ß-actin gene.
|
). When Hep G2 cells
were transfected with a plasmid containing only the apo A-I basal
promoter (-41AI.Luc) and a plasmid containing the hepatic regulatory
region (-256AI.Luc) in front of the luciferase gene, genistein
increased the expression only of the construct containing 256 bp of the
5' region of the apo A-I gene (P = 0.001)
(Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Walsh et al. 1994
). At the molecular level, it has been shown that
estrogen increases the synthesis of apo A-I in liver cells by
increasing the transcription rate of the apo A-I gene (Jin et al. 1998
). This is mediated by the region of the apo A-I
gene that is located between nucleotide -256 and -41, relative to the
start site (Lamon-Fava et al. 1999
). In this report, we
show that the phytoestrogens genistein and daidzein, present in
relatively high amounts in soybeans, share with estrogen the ability to
increase apo A-I secretion from liver cells. Both estrogen and
genistein increased apo A-I gene transcription rate. The mechanism
responsible for this effect of genistein on apo A-I gene expression
is not known, but our results suggest that it is probably mediated by
transcription factors binding to the -256 to -41 region of the apo
A-I gene. This region contains three sequences, A (-214 to -192),
B (-169 to -146), and C (-134 to -119), that have been shown to
bind several liver-enriched and ubiquitous transcription factors
(Harnish et al. 1994
and 1996
, Ladias and Karathanasis 1991
, Rottman et al. 1991
,
Widom et al. 1991
). However, this region does not
contain an estrogen responsive element. Also, electrophoresis mobility
shift experiments have shown that the estrogen receptor (ER) does not
bind to this region (Harnish et al. 1998
). Therefore, it
is unlikely that the observed effect of E2 and
genistein on apo A-I gene expression involves the classic
ER/ligand-mediated regulation of gene expression. In addition, our
results indicate that equal concentrations of E2
and genistein exert a similar increase in apo A-I secretion by Hep
G2 cells. In light of the fact that these compounds have a different
affinity for ER (both 
and ß) (Kuiper et al. 1997
),
this also points to an effect on the apo A-I gene that is not
mediated by the classical ER/ligand genomic activation.
It was shown recently in bovine aortic endothelial cells that
E2 binds to ER localized in the plasma
membrane, where it act as a nongenomic activator of endothelial nitric
oxide production (Kim et al. 1999
). The presence of
estrogen-binding receptors on the surface of cell membranes had
been postulated for some time but their identity had not been
characterized until recently (Nemere and Farach-Carson 1998
). Currently, it is not known whether ER is also localized
in the membrane of liver cells. The ability of the cell membrane ER to
bind E2 and subsequently activate the signal
transduction pathway is a novel function of this receptor and possibly
an important factor for the regulation of gene expression.
It was shown recently in Hep G2 cells that estrogen causes a rapid
increase in inositol triphosphate production through the activation of
the membrane protein kinase C- (Marino et al. 1998
).
It is not know whether this action is mediated by cell membrane ER
receptors; however, since it is abolished by genistein, this does not
seem to be the mechanism involved in apo A-I gene expression.
Genistein is also a tyrosine kinase inhibitor (Akiyama et al. 1987
) and therefore can affect the cell signal transduction
pathway through this specific activity. It is unlikely that the effect
of genistein on apo A-I gene expression is mediated through this
mechanism because E2, which in not known to be an
inhibitor of tyrosine kinase, is able to increase apo A-I
expression to a level similar to genistein.
In conclusion, our in vitro results indicate an effect of phytoestrogens on apo A-I production by liver cells. However, the molecular mechanism that mediates the activation of apo A-I gene expression by both E2 and genistein, and to a smaller extent daidzein, is not currently known. Further studies are required to identify this molecular mechanism. In addition, the effect of these estrogenic compounds on apo A-I production must be tested in animal studies.
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FOOTNOTES |
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2 Abbreviations used: apo, apolipoprotein; CHD, coronary heart disease; DMEM, Dulbecco’s modified Eagle medium; E2, 17-ß-estradiol; ER, estrogen receptor; FBS, fetal bovine serum.
Manuscript received March 9, 2000. Revision accepted June 14, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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