Role of Methoxyestradiols in the Growth Inhibitory Effects of Estradiol on Human
Glomerular Mesangial Cells
Raghvendra K. Dubey, Delbert G. Gillespie, Paul J. Keller, Bruno Imthurn, Lefteris C. Zacharia
and Edwin K. Jackson
Hypertension. 2002;39:418-424
doi: 10.1161/hy0202.103297
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Role of Methoxyestradiols in the Growth Inhibitory Effects
of Estradiol on Human Glomerular Mesangial Cells
Raghvendra K. Dubey, Delbert G. Gillespie, Paul J. Keller, Bruno Imthurn,
Lefteris C. Zacharia, Edwin K. Jackson
Abstract—Metabolism of locally applied 17␤-estradiol (estradiol) to methoxyestradiols contributes to the growth inhibiting
effects of estradiol on vascular smooth muscle cells via an estrogen receptor (ER)-independent mechanism. Because
vascular smooth muscle cells are phenotypically similar to glomerular mesangial cells, it is feasible that estradiol inhibits
glomerular mesangial cell growth via a similar mechanism, and this possibility was investigated. In human glomerular
mesangail cells, estradiol concentration dependently (1 to 100 nmol/L) inhibited serum-induced proliferation (cell
number) and DNA (3[H]-thymidine incorporation) and collagen (3[H]-proline incorporation) synthesis. The inhibitory
effects of estradiol were mimicked by 2-hydroxyestradiol and 2-methoxyestradiol, metabolites of estradiol with little
affinity for ERs. 2-Hydroxyestradiol and 2-methoxyestradiol were more potent growth inhibitors than estradiol. The
inhibitory effects of estradiol were enhanced by CYP450 inducers 3-methylcholanthrene (10 ␮mol/L) and phenobarbital
(10 ␮mol/L) and blocked by the CYP450 inhibitor 1-aminobenzotriazole (10 ␮mol/L). The growth inhibitory effects of
estradiol were also blocked by quercetin (10 ␮mol/L) and OR 486 (10 ␮mol/L) inhibitors of catechol-Omethyltransferase (converts catecholestradiols to methoxyestradiols). ICI182780 (ER antagonist with ER binding
affinity similar to estradiol) blocked the growth inhibitory effects of estradiol (1 to 100 nmol/L) only at concentrations
(⬎50 ␮mol/L) that inhibited estradiol metabolism to catecholestradiols. The growth inhibitory effects of
2-hydroxyestradiol were abrogated by quercetin and OR486 (two structurally dissimilar catechol-O-methyltransferase
inhibitors), but not by ICI182780. However, the growth inhibitory effects of 2-methoxyestradiol were unaltered by
catechol-O-methyltransferase inhibitors and ICI182780. In conclusion, our findings provide the first evidence that
methoxyestradiols mediate the growth inhibitory effects of locally applied estradiol on glomerular mesangial cell growth
via an ER-independent mechanism. (Hypertension. 2002;39[part 2]:418-424.)
Key Words: metabolism 䡲 estrogen 䡲 renal disease 䡲 glomerulosclerosis 䡲 mesangium 䡲 menopause
E
stradiol may induce protective effects on the kidney. For
example, compared with age- matched men, the rate of
progression of renal disease in premenopausal women is
decreased.1,2 With the onset of menopause, decreased synthesis of 17␤-estradiol (estradiol) is accompanied by accelerated
progression of renal diseases, and estradiol replacement
therapy slows this process.1–3
Although estradiol induces renoprotection, the mechanisms
involved remain poorly defined. Inasmuch as coronary artery
disease is the most frequent cause of death in postmenopausal
women,1 most studies have focused on evaluating the effects
of estradiol on vascular cells. In this context, it is well
established that the inhibitory effects of estradiol on vascular
smooth muscle cell growth is known to protect the vasculature against occlusive disorders.1 Analogous to the vascular
remodeling process in atherosclerosis, abnormal growth of
glomerular mesangial cells (GMCs) following glomerular
injury contributes to the glomerular remodeling process
associated with glomerulosclerosis.4 Our previous studies
show that estradiol inhibits GMC growth,5 and we hypothesized that, via this inhibition action, estradiol may protect the
kidney against glomerulosclerosis. However, the mechanisms
by which estradiol inhibits GMC growth are unknown. The
facts that GMCs are phenotypically similar to vascular
smooth muscle cells1,4 and possess functional estrogen receptors1 suggest that the mechanisms by which estradiol induces
its growth inhibitory effects may be similar in GMCs and
vascular smooth muscle cells.
Our recent studies shown that in vascular smooth muscle
cells, the antigrowth effects of estradiol are largely mediated
via its local conversion to hydroxy and methoxy metabolites
that have little affinity for estrogen receptors (ERs).6 This
finding suggests that in addition to the conventional ERdependent mechanisms, ER-independent mechanisms may
Received September 23, 2001; first decision November 7, 2001; accepted November 21, 2001.
From the Center for Clinical Pharmacology, Departments of Medicine (R.G.D., D.G.G., C.Z., E.K.J.), and Pharmacology (C.Z., E.K.J.), University of
Pittsburgh Medical Center, Pittsburgh; and Clinic for Endocrinology Department of Obstetrics and Gynecology, University Hospital Zurich (R.K.D.,
P.J.K., B.I.), Zurich, Switzerland.
Correspondence to Dr Raghvendra K. Dubey, D217, NORD-1, Clinic for Endocrinology, Department of Obstetrics and Gynecology, Frauenklinik,
Zurich 8091, Switzerland. E-mail rag@fhk.usz.ch
© 2002 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
418
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Dubey et al
Figure 1. Schematic representation of the hypothesis and the
experimental approach to demonstrate that metabolism of
estradiol to methoxyestradiols (Methoxy-Es) is responsible for
mediating the inhibitory effects of estradiol on mesangial cell
growth. 2-Methoxyestradiol (2 MeO-E); Catecholestradiols
(Catechol-Es); 2-hydroxyestradiol (2-OH-E); Cytochrome P450
(CYP450); Catechol-O-Methyltransferase (COMT); Inhibition (⫺);
Induction (⫹).
play an important role in inducing the biological effects of
estradiol. This hypothesis is supported by the recent reports
that estradiol prevents injury-induced vascular lesion formation in mice lacking functional ER␣ or ER␤.7,8 Because
GMCs express cytochrome P450 (CYP450) enzymes that can
metabolize estradiol to hydroxyestradiol9 and catechol-Omethyltransferase (COMT)10 that converts hydroxyestradiols
to methoxyestradiols, we hypothesize that the local metabolism of estradiol to hydroxy and methoxy estradiols contributes in part to the antigrowth effects of estradiol on GMCs.
To test our hypothesis, in the present study we compared the
effects of estradiol on fetal calf serum (FCS)-induced growth in
GMCs in the presence and absence of modulators (activators and
inhibitors) of CYP450 and COMT and an ER antagonist (Figure
1). Moreover, we evaluated the capability of GMCs to metabolize 2-hydroxyestradiol to 2-methoxyestradiol and the impact of
this conversion on GMC growth.
Methods
Mesangial Cell Culture
GMCs cultured from normal female donors and in 3rd passage were
obtained from Clonetics Corp. (Walkersville, MD). All chemicals for
cell growth (3H-thymidine incorporation, 3H-proline incorporation,
cell number) studies were purchased from suppliers as described
before.5 GMCs in 3rd passage were grown under standard tissue
culture conditions in phenol red free DMEM/F12 medium supplemented with 10% FCS (steroid free) and antibiotics. Confluent
GMCs were dislodged by trypsinization, washed, and plated for
growth studies at required densities in multiwell plates.
DNA and Collagen Synthesis
3
H-Thymidine and 3H-proline incorporation studies were performed
as measures of DNA and collagen synthesis, respectively. GMCs
were plated at a density of 2.5⫻104 cells/well in 24-well tissue
culture dishes and allowed to grow in DMEM/F12 containing 10%
FCS under standard tissue culture conditions. The monolayers of
GMCs were then growth arrested by feeding DMEM containing
0.4% bovine serum albumin (BSA) for 48 hours. Growth was
stimulated by treating growth arrested GMCs with DMEM supplemented with 2.5% FCS and containing or lacking the various
treatments. For DNA synthesis, after 20 hours of incubation, the cells
were pulsed with 3H-thymidine (1 ␮Ci/mL) for an additional 4
hours. For collagen synthesis, the cells were treated for 48 hours in
Estradiol Inhibits GMC Growth via Metabolites
419
the presence of 3H-L-proline (1 ␮Ci/mL). The experiments were
terminated by washing the cells twice with Dulbecco’s phosphate
buffered saline and twice with ice-cold trichloroacetic acid (10%).
The precipitate was solubilized in 500 ␮L of 0.3N NaOH and 0.1%
SDS after incubation at 50°C for 2 hours. Aliquots from 4 wells for
each treatment with 10 mL scintillation fluid were counted in a liquid
scintillation counter, and each experiment was conducted using three
to four separate cultures. 3H-Thymidine incorporation studies were
conducted in subconfluent monolayers. However, to ensure that
changes in collagen synthesis were not due to a decreases in cell
number, 3H-proline incorporation studies were conducted in confluent monolayers of cells in which changes in cell number were
precluded. Cell counting was performed in cells treated in parallel to
the cells used for the collagen synthesis, and the data were normalized to cell number.
Cell Proliferation
Cell counting was performed as a direct measure of cell proliferation.
Trypsinized GMCs were suspended in DMEM/F12 containing 10%
FCS and plated in a 24-well culture dish at a density of 1⫻104
cells/well. After incubation for 24 hours, cells were growth arrested
by feeding DMEM containing 0.4% BSA for 48 hours. GMCs were
then treated every 24 hours for 2 to 12 days with DMEM supplemented with 2.5% FCS and containing or lacking various treatments.
The treatments were terminated on day 2, 4, 8, or 12 and cells
dislodged with trypsin-EDTA, diluted in Isoton-II, and counted with
a Coulter counter. Aliquots from three wells were counted for each
group using three separate cultures.
Protein Determination
Total cellular protein was determined by the Bio-Rad detergent
method, which uses a modification of the Lowry assay with BSA as
a standard.
Metabolism of Catecholestradiols to Methoxyestradiols
Confluent GMCs were incubated with 2-hydroxyestradiol for 2
hours, internal standard (16␣-hydroxyestradiol) was added, samples
were extracted with methylene chloride, extracts were dried under
vacuum, residues were reconstituted in mobile phase, and samples
were analyzed by high-performance liquid chromatography with
ultraviolet detection using gradient elution, as previously descibed.11
Statistics
Results are expressed as mean⫾SEM. Statistical analysis was
performed with the use of Student’s unpaired t test and one way
ANOVA. Values of P⬍0.05 are considered to be significantly
different.
Results
Treatment with FCS stimulated 3H-thymidine and 3H-proline
incorporation and cell number by approximately 5- to 7-fold
(P⬍0.05). To address the potential role of endogenous
estradiol metabolites in regulating GMC growth, we first
tested the potency of estradiol and estradiol metabolites to
inhibit FCS-induced growth on GMCs. The 2-hydroxy and
2-methoxy metabolites of estradiol inhibited FCS-induced
DNA synthesis (Figure 2A), proliferation (Figure 2B) and
collagen synthesis (Figure 2C) in the following order of
potency: 2-methoxyestradiol⬎2-hydroxyestradiol⬎estradiol.
In contrast, estrone, estriol, 16␣-hydroxyestrone, 2-hydroxyestrone, estrone sulfate, and 4-methoxyestrone were
significantly less potent and did not inhibit GMC growth
(DNA synthesis, collagen synthesis, cell proliferation) within
the concentration range (1 to 100 nmol/L) used (data not
shown). The lowest concentration of estradiol,
2-methoxyestradiol, and 2-hydroxyestradiol that significantly
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February 2002 Part II
Figure 2. Effects of increasing concentrations of estradiol,
2-hydroxyestradiol, and 2-methoxyestradiol on 2.5% FCSinduced DNA incorporation after 24 hours (A), collagen synthesis after 48 hrs (B), and cell number after 4 days (C) in human
GMCs. The results are presented as percent change from control (GMCs treated with FCS alone). Values for each data point
represent means⫾SEM from 3 separate experiments conducted
in quadruplicate.
inhibited FCS-induced increases in cell number was 1
nmol/L. Treatment of GMCs for 4 days with a physiological
concentration (1 nmol/L) of estradiol inhibited cell proliferation by 16%. At this concentration, 2-methoxyestradiol and
2-hydroxyestradiol inhibited cell number by 38% and 28%,
respectively.
To investigate whether the local metabolism of estradiol to
metabolites by CYP450s is responsible for the growth inhibitory
effects of estradiol, we studied the effects of estradiol in the
presence and absence of inducers (3-methylcholantherene and
phenobarbital6,12 and an inhibitor (1-aminobenzotriazole13) of
CYP450s. Treatment of GMCs for 48 hours with
3-methylcholantherene (10 ␮mol/L), phenobarbital (10 ␮mol/
L), or 1-aminobenzotriazole did not influence FCS-induced
DNA synthesis, cell proliferation, or collagen synthesis. The
inhibitory effects of physiological concentrations of estradiol (1
nmol/L) on FCS-induced DNA synthesis and cell proliferation
were enhanced by 3-methylcholantherene and phenobarbital
(Figures 3A and 3B). Similar modulatory effects of
3-methylcholantherene and phenobarbital were also observed on
collagen synthesis (Figure 3C). The inhibitory effects of estradiol (1 nmol/L) on FCS-induced proliferation of GMCs were
enhanced from 18% to 41%, and 35% by 3-methylcholantherene
and phenobarbital, respectively. In the presence of phenobarbital
and 3-methylcholantherene, the concentration-dependent inhibitory effects of estradiol on FCS-induced DNA synthesis, cell
number, and collagen synthesis were almost doubled and the
inhibitory curve shifted to the left (Figure 3). In contrast to the
CYP450 inducers, the concentration-dependent inhibitory effects of estradiol on cell proliferation, DNA synthesis, and
collagen synthesis were abolished by the CYP450 inhibitor
Figure 3. Modulatory effects of CYP450 inducers
3-methylcholantherene and phenobarbital on the concentrationdependent inhibitory effects of estradiol (␤-Est; 1 to 100 nmol/L) on
DNA synthesis after 24 hours (A), collagen synthesis after 48 hours
(B), and cell number after 4 days (C). For 3-methylcholantherene
and phenobarbital (PB), the concentration was 10 ␮mol/L. Values
for each data point represent means⫾SEM from 3 separate experiments conducted in quadruplicate. *P⬍0.05 versus cells treated
with FCS alone; §significantly (P⬍0.05) different from estradiol
alone.
1-aminobenzotriazole (Figure 4). Moreover, the enhanced inhibitory effects of estradiol observed in the presence of
3-methylcholantherene plus phenobarbital on all parameters of
GMC growth were also blocked by 1-aminobenzotriazole (Figure 4). Trypan blue exclusion tests and MTT assay indicated no
loss in viability of cells treated with various agents.
The concentration-dependent inhibitory effects of estradiol
on cell proliferation, DNA synthesis, and collagen synthesis
were abolished by the catechol-O-methyltransferase (COMT)
inhibitors quercetin and OR48614 (Figure 5). Moreover, the
enhanced inhibitory effects of estradiol observed in the
presence of CYP450 inducers 3-methylcholantherene plus
phenobarbital on all parameters of GMC growth were also
blocked by quercetin and OR486 (data not shown).
The inhibitory effects of 2-hydroxyestradiol, but not
2-methoxyestradiol, on DNA synthesis (Figure 6A), collagen
synthesis (Figure 6B), and proliferation (Figure 6C) were
completely prevented by 10 ␮mol/L quercetin and OR486. In
contrast to quercetin and OR486, ICI182780 (50 ␮mol/L), an
estrogen receptor antagonist,6 did not block the growth
inhibitory effects of either 2-hydroxyestradiol or
2-methoxyestradiol (Figures 6A to 6C).
The growth inhibitory effects of estradiol were blocked by
ICI182780, and these effects were concentration dependent
(Figure 7A). The lowest concentration of ICI182780 that
significantly blocked the inhibitory effects of 100 nmol/L
estradiol was 10 ␮mol/L, and a concentration of 50 ␮mol/L
ICI182780 completely blocked the inhibitory effects of 100
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Dubey et al
Figure 4. Modulatory effects of the CYP450 inhibitor
1-aminobenzotriazole (ABT, 10 ␮mol/L) on the concentrationdependent inhibitory effects of estradiol (␤-E; 1 to 100 nmol/L)
on 2.5% FCs-induced DNA synthesis after 24 hours (top), collagen synthesis after 48 hours (middle), and cell number after 4
days (bottom) in GMCs simultaneously treated with or without
3-methylcholantherene (3 MC; 10 ␮mol/L) plus phenobarbital
(PB; 10 ␮mol/L). Values for each data point represent
means⫾SEM from 3 separate experiments conducted in quadruplicate. *P⬍0.05 versus cells treated with FCS alone; §significantly (P⬍0.05) different from estradiol alone.
nmol/L estradiol (Figure 7A). Compared with ICI182780,
both 1-aminobenzotriazole and quercetin were more potent in
antagonizing the growth inhibitory effects of estradiol (Figure
7A). Quercetin is not only a COMT substrate, but also a
Estradiol Inhibits GMC Growth via Metabolites
421
Figure 6. Inhibitory effects of 2-hydroxyestradiol (OE; 0.1
␮mol/L) and 2-methoxyestradiol (ME; 0.1 ␮mol/L) on 2.5% FCSinduced growth (DNA synthesis after 24 hours, top; cell number
after 4 days, middle; and collagen synthesis after 48 hours, bottom) of human GMCs in the presence and absence of the estrogen receptor antagonist ICI182780 (ICI; 50 ␮mol/L), quercetin
(Quer; 10 ␮mol/L), or OR486 (OR; 10 ␮mol/L). Values are
mean⫾SEM from three separate experiments conducted in quadruplicate. *P⬍0.05 versus control; §significant reversal of inhibitory effect.
ligand for type II ER. To rule out the participation of the type
II ER in mediating the modulatory effects of quercetin on the
growth effects of estradiol, we evaluated the effects of
estradiol on GMC growth in the presence of luteolin, a high
affinity type II ER ligand.6 In contrast to quercetin and
OR486, the inhibitory effects of estradiol on GMC growth
were not blocked by luteolin (Figure 7A).
We have previously shown that at concentrations greater
than 1 ␮mol/L, ICI182780 inhibits the metabolism of estradiol to 2- and 4-hydroxyestradiol by CYP1A2 and with
apparent Kis of 45 ␮mmol/L and 27 ␮mol/L, respectively.6 In
the present study, 1 ␮mol/L ICI 182780 was unable to block
the inhibitory effects of 1 nmol/L estradiol, even though the
estradiol to ICI182780 ratio was 1:1000 (Figure 7B). Moreover, the inhibitory effects of physiological concentrations of
estradiol on GMC growth were cumulative in nature, ie, the
inhibitory effects of estradiol increased with time of exposure. Treatment of GMCs with a physiological concentration
(1 nmol/L) of estradiol for 2, 4, 8, and 12 days inhibited
FCS-induced cell proliferation by 7⫾1%, 20⫾2%, 32⫾2%,
and 44⫾3%, respectively (Figure 7B).
GMCs metabolized 2-hydroxyestradiol (2 ␮mol/L for 1 hour)
to 2-methoxyestradiol, and this metabolism was inhibited by 10
␮mol/L of quercetin (96⫾0.8%, P⬍0.05) or OR486 (95⫾0.5%,
P⬍0.05) but not by ICI182780 (Figure 7C).
Discussion
Figure 5. Modulatory effects of the COMT inhibitors, quercetin
(Quer) and OR4863 (OR) on the concentration-dependent inhibitory effects of estradiol (␤-Est;1 to 100 nmol/L) on DNA synthesis after 24 hours (A), collagen synthesis after 48 hours (B), and
cell number after 4 days (C). The concentrations for quercetin
and OR486 were 10 ␮mol/L. Values for each data point represent means⫾SEM from 3 separate experiments conducted in
quadruplicate. *P⬍0.05 versus cells treated with FCS alone;
§significantly (P⬍0.05) different from estradiol alone.
Our findings strongly suggest that the inhibitory effects of
estradiol on GMC growth are mediated via CYP450-derived
metabolites. Treatment of GMCs with estradiol,
2-hydroxyestradiol, or 2-methoxyestradiol, but not estrone,
estriol, 16 ␣ -hydroxyestradiols, estrone sulfate, hydroxyestrone, or methoxyestrone, inhibits serum-induced
GMC growth, and 2-hydroxyestradiol or 2-methoxyestradiol
are more potent than estradiol in this regard. Importantly,
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February 2002 Part II
Figure 7. A, Concentration-response curve comparing the abrogatory effects of 1-aminobenzotriazole (ABT), quercetin,
ICI182780 (ICI), and luteolin on the inhibitory effects of estradiol
(0.1 ␮mol/L) on 2.5% FCS-induced DNA synthesis in GMCs
treated for 24 hours. *P⬍0.05 versus GMCs treated with FCS
alone. B, Antagonistic effects of concentrations of ICI182780
(ICI) that inhibit estradiol (␤E) metabolism (50 ␮mol/L) and do
not inhibit estradiol metabolism (1 ␮mol/L) on the inhibitory
effects of 1 and 50 nmol/L estradiol, respectively, on FCSinduced growth (cell number) of GMCs treated for 2, 4, 8, or 12
days. The ratio of estradiol and ICI182780 was 1:1000 under
both treatment conditions. The data are presented as percent of
control where 100% is defined as the increase in cell number in
response to 2.5% FCS alone. * P⬍0.05 versus GMCs treated
with FCS alone; § significant (P⬍0.05) reversal of the inhibitory
effects of estradiol. (C) Inhibitory effects of quercetin (Que; 10
␮mol/L), ICI182780 (ICI; 50 ␮mol/L), and OR486 (OR; 10
␮mol/L), on the metabolism of 2-hydroxyestradiol (2 ␮mol/L) to
2-methoxyestradiol (2 ME) by cultured GMCs. *P⬍0.05 versus
methoxyestradiol (2-ME) formation by GMCs alone.
3-methylcholantherene and phenobarbital, CYP450 inducers12 with no affinity for ERs, enhanced the inhibitory effects
of estradiol. Moreover, 1-aminobenzotriazole, a broad spectrum CYP450 inhibitor13 with no affinity for ERs,6 abrogated
the antigrowth effects of estradiol both in the presence and
absence of CYP450 inducers (see schematic representation in
Figure 1). Our conclusion that the inhibitory effects of
estradiol on GMC growth are mediated via CYP450-derived
metabolites is further supported by the well-established findings that the CYP1A1 and CYP1B1, isozymes of CYP450
responsible for the metabolism of estradiol to catecholestradiols, are highly expressed in GMCs.9
The facts that catecholestradiols are rapidly metabolized to
methoxyestradiols by COMT,14 GMCs express COMT activity 10 , and 2-methoxyestradiol is more potent than
2-hydroxyestradiol in inhibiting GMC growth lead us to
hypothesize that methoxyestradiols are the ultimate mediators
of the ER-independent antigrowth effects of estradiol. Our
hypothesis is supported by the observations that the COMT
inhibitors quercetin and OR486,14 which have no binding
affinity for ERs,6 attenuate the inhibitory effects of
2-hydroxyestradiol, but not 2-methoxyestradiol, on GMC
growth. Moreover, quercetin, as well as OR486, block the
growth inhibitory effects of estradiol, either in the absence or
presence of CPY450 inducers. In contrast, ICI182780 (50
␮mol/L), an ER antagonist,6 does not block the growth
inhibitory effects of either 2-hydroxyestradiol or
2-methoxyestradiol. These findings provide evidence that the
conversion of 2-hydroxyestradiol to 2-methoxyestradiol by
COMT is responsible for the inhibitory effects of
2-hydroxyestradiol, and that the effects of 2-methoxyestradiol
are ER-independent, as would be expected by the low affinity
of 2-hydroxyestradiol and 2-methoxyestradiol for ERs. The
hypothesis that the inhibitory effects of estradiol are mediated
via generation of methoxyestradiols is further supported by
our observation that GMCs metabolize 2-hydroxyestradiol to
2-methoxyestradiol and that this metabolic conversion is
blocked by the COMT inhibitors quercetin and OR486.
Quercetin not only blocks the conversion of catecholestrogens to methoxyestrogens,11 but also binds to the type II ER15
that has been implicated in regulating cell growth.15 The
finding that the inhibitory effects of estradiol are not blocked
by luteolin, a high affinity type II ER ligand,15 rules out the
participation of type II ER. Moreover, it supports the conclusion that quercetin blocks the inhibitory effects of estradiol by
inhibiting COMT and blocking the formation of methoxyestradiols. This contention is directly supported by the
observation that OR486, an established COMT inhibitor,
blocks the antigrowth effects of both estradiol and
2-hydroxyestradiol, but not 2-methoxyestradiol.
The growth inhibitory effects of estradiol are blocked by
ICI182780, an ER antagonist that binds with equal affinity to
both ER␣ and ER␤,16 a finding seemingly inconsistent with
the hypothesis that methoxyestradiols mediate the growth
inhibitory effects of estradiol. However, because ICI 182780
is chemically similar to estradiol, it is feasible that it not only
binds to ERs but also competes with estradiol for CYP450s
and inhibits estradiol metabolism. This notion is supported by
our recent finding that ICI182780 inhibits the metabolism of
estradiol to catecholestradiols in extracts of human hepG2
cells expressing the CYP1A2 isozyme, which is responsible
for metabolizing estradiol to catecholestradiols.6 The potential that ICI182780 may block the antigrowth effects of
estradiol by inhibiting COMT can also be ruled out because
ICI182780 blocks the antigrowth effects of estradiol, but not
2-hydroxyestradiol and 2-methoxyestradiol. Moreover, in
contrast to quercetin and OR486, ICI182780 fails to inhibit
the conversion of 2-hydroxyestradiol to 2-methoxyestradiol.
The above findings suggest that the inhibitory effects of
ICI182780 may be mediated either via antagonism of ER or
via inhibition of estradiol metabolism. However, the fact that
the antagonistic effects of ICI182780 are not dependent on
the estradiol to ICI182780 ratio, but rather on concentrations
of ICI182780 that inhibit estradiol metabolism,6 suggests that
ICI182780 blocks the inhibitory effects of estradiol by
blocking estradiol metabolism to catecholestradiols, the precursors of methoxyestradiols.
Our contention that local conversion of estradiol to methoxyestradiols is responsible for its ER-independent antigrowth effects in GMCs is supported by our recent finding
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Dubey et al
that local metabolism of estradiol to methoxyestradiol inhibits the growth of vascular smooth muscle cells, which are
phenotypically similar to GMCs.6 Moreover, our recent
studies support the participation of this mechanism in inducing the antigrowth effects of estradiol in cardiac fibroblasts.17
Taken together, our findings suggest that the conversion of
estradiol to 2-methoxyestradiol may be a physiologically
relevant and a prominent pathway via which estradiol regulates cell growth. In this context, it is important to note that
metabolism of estradiol to methoxyestradiols plays a role in
regulating growth of tumor/cancer cells (mammary tumors,
kidney tumors in Syrian hamsters, and endometrial cancer).18
In vivo studies provide evidence that decreased formation of
2-methoxyestradiol and its precursor, 2-hydroxyestradiol, is
associated with mammary cancer, endometrial cancer, and
renal tumors in Syrian hamters.18
In contrast to our findings, an earlier study showed that
physiological concentrations of estradiol induced DNA synthesis and proliferation in GMCs that were not growth
arrested.19 The disparate effects of estradiol in the two studies
may be due to the culture conditions. In this regard, estradiol
induces MAP kinase activity in mesangial cells that are not
growth arrested, whereas in growth arrested (serum starved)
GMCs, estradiol has no effect on basal MAP kinase activity
and inhibits mitogen (PDGF and Ang II)-induced MAP
kinase activity.20 Because in growth arrested GMCs mitogens
induce cell proliferation via activation of MAP kinase activity21, and because estradiol inhibits these effects, it is feasible
that the growth inhibitory effects of estradiol on GMCs may
depend on whether a growth stimulus is present and whether
the GMCs are syncronized in G0 phase of the cell cycle.
Additional studies are required to resolve the discrepancy
between these two studies.
With regard to the renal system, our finding that estradiol
metabolism to methoxyestradiols is responsible for mediating
the growth inhibiting effects of locally applied estradiol on
GMCs may have clinical implications. Since increased proliferation of GMCs plays a key role in glomerulosclerosis,1
estradiol metabolites may protect against glomerular remodeling by inhibiting cell growth. Thus, the protective effects of
estradiol on the progression of renal disease in postmenopausal women may not only be dependent on estradiol levels,
but also on the capability of the individual to metabolize
estradiol to these metabolites. However, future studies are
required to investigate this possibility in more detail.
The finding that conversion of 2-hydroxyestradiol to
2-methoxyestradiol by COMT is essential for mediating the
antigrowth effects of estradiol has additional implications.
Apart from metabolizing catecholestradiols, COMT, which is
highly expressed in both cardiovascular11,12 and renal
cells,10,14 is also involved in the metabolism of catecholamines,10,11,14 which are known to induce deleterious effects
on the renal system.4 Because both catecholestradiols and
catecholamines share COMT for their metabolism, interactions of these compounds at COMT may play an important
role in determining the effects of these molecules on the renal
system. This contention is supported by our recent findings
that catecholamines block the antigrowth effects of estradiol
and 2-hydroxyestradiol on rat GMCs,10 and that catechol-
Estradiol Inhibits GMC Growth via Metabolites
423
amines inhibit the conversion of 2-hydroxyestradiol to
2-methoxyestradiol by rat GMCs.10 In the same context, we
have recently reported that in vascular smooth muscle cells,
norepinephrine, epinephrine, and isoproterenol inhibit the
metabolism of 2-hydroxyestradiol to 2-methoxyestradiol and
abrogate the antigrowth effects of 2-hydroxyestradiol on
vascular smooth muscle cells.22 Therefore, interactions between catecholestradiols and catecholamines may play an
important role in determining the effects of estradiol on the
kidney.
In summary, our findings provide the first evidence that the
antigrowth effects of estradiol on human GMCs are mediated
via an ER-independent pathway that involves the local
conversion of estradiol to methoxyestradiols (Figure 1), that
estradiol may protect against progression of renal disease by
inhibitng GMC growth, and that estradiol metabolism may be
an important determinant of the renal protective effects of
estradiol. Thus, interindividual differences, either genetic or
acquired, in estradiol metabolism may define a given female’s risk of renal disease and influence the renal benefit she
receives from estradiol replacement therapy in the postmenopausal state. These findings also imply that nonfeminizing
estradiol metabolites may confer renal protection in both
women and men.
Acknowledgments
This study was supported by Swiss National Science Foundation
grants 32–54172.98 and 32-640.00 and by the National Institutes of
Health grant HL55314.
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