Nephrology Division, Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York 10467
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ABSTRACT |
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Estrogen receptor modulators (SERMs) are "designer
drugs" that exert estrogen-like actions in some cells but not in
others. We examined the effects of the SERMs LY-117018 (an analog of
raloxifene) and tamoxifen on mesangial cells synthesis of type I and
type IV collagen. We found that LY-117018 and tamoxifen suppressed mesangial cell type IV collagen gene transcription and type IV collagen
protein synthesis in a dose-dependent manner, with a potency identical
to that of estradiol. Type I collagen synthesis was also suppressed by
LY-117018 in a dose-dependent manner with a potency identical to that
of estradiol but greater than that of tamoxifen. Genistein, which
selectively binds to estrogen receptor- in nanomolar concentrations,
suppressed type I and type IV collagen synthesis, suggesting that
estrogen receptor-
mediates the effects of estrogen on collagen
synthesis. Because matrix accumulation is central to the development of
glomerulosclerosis, second-generation SERMs may prove clinically useful
in ameliorating progressive renal disease without the adverse effects
of estrogen on reproductive tissues.
estradiol; angiotensin II; transforming growth
factor-1; endothelin
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INTRODUCTION |
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SELECTIVE ESTROGEN RECEPTOR modulators, SERMs, are compounds that bind to the estrogen receptor but have tissue-specific effects that may differ from those of estrogen itself (4, 12, 22). The promise of the SERMs lies in their ability to reproduce the beneficial effects of estrogen on bone, endothelium, and lipoprotein metabolism without reproducing the undesirable effects of estrogen on reproductive tissues (4, 12, 22). Raloxifene, a second-generation SERM, has a more favorable tissue selectivity profile than tamoxifen, a first-generation SERM (4, 12, 22). Although both compounds act as estrogen antagonists in mammary tissue, tamoxifen is a partial estrogen agonist in endometrial tissue whereas raloxifene is a complete antagonist in the uterus (4).
Renal disease progresses less rapidly in women than in men (24, 33). In the course of investigating the mechanisms responsible for this observation, we found that estradiol suppresses the synthesis of types I and IV collagen by mesangial cells grown in the presence of serum (18, 19, 25, 26, 31, 32). The ability of estradiol to suppress mesangial cell collagen synthesis may reduce matrix accumulation after glomerular injury and contribute to the beneficial effect of female gender on the progression of renal disease (33).
We reasoned that if second-generation selective estrogen receptor
modulators reproduce estradiol's suppressive effects on mesangial cell
collagen synthesis, they might prove clinically useful in ameliorating
progressive glomerosclerosis in chronic renal disease without
reproducing the adverse effects of estradiol on reproductive tissue.
The potential clinical usefulness of second-generations SERMs in
limiting progressive renal disease led us to explore in detail their
antifibrotic actions. In the present study we examined the effects of a
first-generation SERM (tamoxifen) and a second-generation SERM
(LY-117018) on the synthesis of type I and type IV collagen by cultured
mesangial cells. We also investigated the effects of the SERMs on the
ability of fibrogenic agents such as transforming growth factor-1
(TGF-
1), ANG II, and endothelin-1 (ET-1) to stimulate mesangial cell
type
1(IV) gene (COL4A1) transcription and type IV
collagen synthesis.
We found that LY-117018 and tamoxifen suppress COL4A1 gene transcription and type IV collagen protein synthesis in a dose-dependent manner, with a potency identical to that of estradiol. Type I collagen synthesis was also suppressed by LY-117018 in a dose-dependent manner with a potency identical to that of estradiol but greater than that of tamoxifen.
We also examined the effect of the SERMs on the ability of the
fibrogenic agents TGF-1, ANG II, and ET-1 to stimulate COL4A1 gene
transcription and type IV collagen synthesis. Estradiol, in
physiological concentrations, reversed the stimulatory effects of ANG
II and ET-1 on COL4A1 gene transcription and type IV collagen synthesis
by antagonizing the actions of TGF-
1. The SERMs reproduced the
effects of estradiol, reversing the actions of ANG II and ET-1 on type
IV collagen via antagonism of the autocrine/paracrine effects of
TGF-
1.
Last, we demonstrated that estrogen receptor- (ER-
) and estrogen
receptor-
(ER-
) are present in murine mesangial cells and that
the effects of estrogen on collagen synthesis are mediated, at least in
part, by ER-
.
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METHODS |
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Isolation and characterization of murine mesangial cells. Mesangial cells were isolated from kidneys of 8- to 10-wk-old naive SJL/J (H-2S) mice by differential glomerular sieving. The present studies were performed in an immortalized, differentiated murine mesangial cell line transformed with nonreplicating, non-capsid-forming SV40 virus (strain Rh 911). The cells express receptors for ANG II and stain positive for Thy-1 antigen, desmin, vimentin, and types I and IV collagens but fail to bind antibody directed against a proximal tubular antigen (36). To exclude the possibility that our results were influenced by SV40 transformation per se, all experiments (except reporter gene studies) were reproduced in nontransformed male murine mesangial cells. Because identical results were obtained in transformed and nontransformed cells, all data were combined.
Construction of reporter gene plasmids.
A plasmid containing portions of the gene encoding murine
1(IV)-collagen was linked to luciferase coding sequences
to form a reporter construct, HB35, has previously been described
(8). A 2.3-kb Xho I fragment derived from p184
(gift of Dr. P. Killen, University of Michigan) (5,
13) spanning the first three exons, the first two introns,
and a portion of the third intron of the
2(IV)-collagen
gene, the intergenic bidirectional promoter region, and a portion of
the
1(IV)-collagen transcription unit was cloned into
pBluescript (Stratagene, La Jolla, CA) and was truncated within the
third intron of the
2(IV) gene by excising the
appropriate fragment with Hind III. The fragment was then ligated into the Hind III site of the luciferase expression plasmid, pSVOAlucD5' (7), and its orientation was determined by
restriction mapping (17). An additional 3.2-kb fragment of
the first intron of the
1(IV) gene previously shown to
modulate the activity of the
1(IV) promoter
(5, 13), corresponding to a BamH
I-linked 3.2-kb Xba fragment of the first intron of
1(IV), was inserted into the BamH I site
downstream from the 3' end of the luciferase coding sequences following
partial BamH I digestion (17). Intron fragment
orientation and position were confirmed by restriction mapping
(17).
Stable transfection. Murine mesangial cells were cotransfected with HB35 and a selection plasmid pSV2-Neo encoding for neomycin-resistance at a molar ratio of 10:1 by using the CaPO4-DNA precipitation procedure (2). Cells were then grown in selection medium containing Geneticin (G-418). Cells surviving in medium with Geneticin were expanded and reselected with the neomycin analog. Stable transfectants exhibited patterns of growth behavior and protein synthesis similar to the parent cell line (8).
Luciferase assay.
Cells were plated in six-well plates and grown in phenol-free,
serum-free RPMI. In some experiments cells were exposed to 17-estradiol (Sigma Chemical, St. Louis, MO); LY-117018 (a
second-generation SERM analog of raloxifene; Lily Research
Laboratories, Indianapolis, IN); tamoxifen (a first-generation SERM;
Sigma Chemical), and genistein (a phytoestrogen; Sigma Chemical);
TGF-
1 (R & D Systems, Minneapolis, MN); ANG II (Sigma Chemical);
ET-1 (Sigma Chemcial); ANG II antipeptide (a nonselective ANG II
receptor antagonist; Sigma Chemical); neutralizing antibody to TGF-
1
(R&D Systems); nonimmune IgG (Sigma Chemical); or PD 0156707 (a
selective ETA receptor antagonist; Parke-Davis
Pharmaceutical Research, Ann Arbor, MI), alone or in combination, for
24 h. Cells were washed twice with PBS, then lysed with 100 µl
Reporter Lysis Buffer (Promega, Madison, WI) at room temperature for 15 min. Wells were then scraped, and the cell lysate was transferred to a
microcentrifuge tube and placed on ice. Tubes were vortexed and
microcentrifuged for 2 min at 4°C. The suspension was transferred to
a new microcentrifuge tube and stored at
70°C until assayed. Twenty
microliters of cell extract were mixed with 10 µl of assay reagent
[20 mM tricine, 1.07 mM (MgCO3)4
Mg(OH)2 · 5H2O, 2.67 mM
MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol (DTT), 270 µM
coenzyme A, 470 µM luciferin, 530 µM ATP, pH 7.8] at room
temperature (all reagents were from Sigma Chemical, St. Louis, MO).
Light emission was measured directly at room temperature over a 10-s
period in a luminometer (Promega). Blank reactions were determined with
equivalent volumes of lysis buffer substituted for cell lysates, and
these values were subtracted from experimental values. Luciferase
activity was expressed per milligram of cellular protein in the
supernatant as determined by the Bio-Rad protein assay (Bio-Rad,
Richmond, CA). Relative luciferase units were calculated as percentages
of control values, where 100% is the value obtained with control media
(serum-free, no added agents).
Western blotting.
In studies utilizing TGF-1, ANG II, or ET-1, cells were grown
in phenol-free, serum-free RPMI as described above. In all other
studies, cells were grown in phenol-free RPMI supplemented with 20%
FCS. Cells were not growth arrested before study. Media was collected
and concentrated to an identical final volume by using an Amicon
Centriprep-10 concentrator (Grace, Beverly, MA). Protein content was
measured in a 0.1-ml aliquot of the concentrated media (Bio-Rad Protein
Assay, Richmond, CA). To prepare the samples for SDS-PAGE, 1 ml of 10%
trichloroacetic acid was added to 4 mg protein of concentrated media,
and the final volume was brought to 2 ml with distilled water. The
sample was vortexed and then centrifuged at 2,000 rpm for 10 min. The
pellet was dissolved in 1 ml of loading buffer (2% SDS, 10% glycerol,
50 nM Tris, 3% bromophenol blue, 2%
- mercaptoethanol, pH 7.6),
boiled for 5 min, then immediately placed on ice. Samples (25, 50, 75 or 100 µg) were loaded for electrophoretic separation of proteins.
SDS-PAGE was performed by standard techniques, and proteins were
transferred to a polyvinylidene difluoride microporous membrane.
Preparation of nuclear extracts. Mesangial cells were scraped into PBS and pelleted by centrifuging at 3,000 g for 10 min at 4°C (30). The cells were resuspended in hypotonic buffer [(in mM) 10 HEPES, pH 7.9, 1.5 MgCl2, 10 KCl, 0.2 phenylmethylsulfonyl fluoride (PMSF), 0.5 DTT (Sigma Chemical)], recentrifuged at 3,000 g for 5 min, resuspended in the hypotonic buffer, and allowed to swell for 10 min on ice. The cells were homogenized in a glass Dounce homogenizer. Nuclei were collected by centrifuging at 3,300 g for 15 min. The nuclear pellet was resuspended in low-salt buffer [20 mM HEPES, 2% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT (Sigma Chemical)]. With gentle stirring, a volume of high-salt buffer (1.2 M KCl) equal to one-half the packed nuclear volume was added dropwise. The protein concentration was measured by colorimetric assay (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assay.
Four micrograms of nuclear extracts were mixed with 2 mg of poly
(dI:dC) in 20 µl of a reaction buffer consisting of 25 mM HEPES, pH
7.5, 1.2 mM DTT, 4 mM MgCl2, 150 mM NaCl, 5% glycerol, 0.005% bromphenol blue, and 0.05% Nonidet P-40 (Sigma Chemical) (30). The mixture was incubated on ice for 15 min,
followed by addition of 10 fmo1 of 32P end-labeled activaor
protein-1 (AP-1) consensus binding sequence oligonucleotide (nucleotide
sequence: 5'-CGCTTGATGAGTCAGCCGGAA-3'; Promega). Incubation was
continued for 30 min. The incubation mixture was subjected to
electrophoresis on a 6% polyacrylamide gel in Tris-glycine buffer. The
gels were dried, and autoradiography was performed at 70°C with an
intensifying screen. Bands were quantitated by laser densitometry
(model 300S, Molecular Dynamics). Competition experiments were
performed with a 200-fold excess of unlabeled AP-1 consensus binding
sequence oligonucleotide.
Statistics. For each individual data point, the mean of at least three to seven experiments (using 3 replicate wells/experiment) was calculated. The data are means ± SE. Differences among groups were tested by analysis of variance with Scheffé's correction. P < 0.05 was considered a significant difference.
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RESULTS |
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Identification of ERs in mesangial cells.
We used a mouse monoclonal anti-ER- antibody that did not
cross-react with ER-
to demonstrate the presence of ER-
in murine mesangial cell extracts by Western blotting (Fig.
1A, top). We used a
rabbit polyclonal anti-ER-
antibody that did not cross-react with
ER-
to demonstrate the presence of ER-
in murine mesangial cell
extracts by Western blotting (Fig. 1A, bottom).
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SERMs suppress mesangial cell type IV collagen gene transcription
and protein synthesis.
The SERM LY-117018 suppressed COL4A1 gene transcription in a
dose-dependent manner in murine mesangial cells grown in 20% FCS (Fig.
1B). At a LY-117018 concentration of 107 M,
LY-117018 decreased COL4A1 gene transcription by 31.4%
(P < 0.0001 vs. control). At a concentration of
LY-117018 equivalent to physiological concentrations of estradiol in
premenopausal women (10
9 M), COL4A1 gene transcription
was reduced by 16.4% (P < 0.0001 vs. control). The
potency of LY-117018 was similar to that of estradiol and tamoxifen at
identical concentrations [10
9 M; LY-117018: 83.7 ± 1.7, expressed as a percentage of control values; estradiol: 82.3 ± 1.0; tamoxifen: 89.1 ± 1.8; not significant (NS) vs. LY-117018
and P NS vs. estradiol (Fig. 1B)]. The
suppressive effects of LY-117018, tamoxifen, and estradiol on COL4A1
gene transcription were not additive.
SERMs suppress mesangial cell type I collagen synthesis.
LY-117018 suppressed type I collagen synthesis by murine mesangial
cells in a dose-dependent manner (Fig.
2A). Type I collagen synthesis
was reduced by 75.7% at an LY-117018 concentration of 107 M (P < 0.0001 vs. control) and by
47.8% at a LY-117018 concentration of 10
9 M
(P < 0.0001 vs. control). The potency of LY-117018 was
similar to that of estradiol but greater than that of tamoxifen at
identical concentrations (10
9 M; estradiol: 37.3 ± 6.9, P < 0.002 vs. control; LY-117018: 36.4 ± 8.3, expressed as a percentage of control values, P < 0.009 vs. control, P NS vs estradiol; tamoxifen: 68.6 ± 10.7, P NS vs. control, P < 0.05 vs.
LY-117018) (Fig. 2B).
|
ER- mediates the suppressive effects of estrogen on collagen
synthesis.
Genistein, which selectively binds to ER-
in nanomolar
concentrations, suppressed type I and type IV collagen synthesis in a
dose-dependent manner (10
9 to 10
7 M) (Fig.
2C). The effects of genistein (10
9 M) were
reversed by ICI-182780 (10
8 M) (Fig. 2C).
SERMs upregulate MAP kinase and AP-1 activity.
We have previously shown that estradiol suppresses mesangial cell type
I collagen synthesis by increasing the activity of AP-1 via
upregulation of the mitogen-activated protein kinase pathway
(25, 31). We examined the hypothesis that the
SERMs suppress type I collagen synthesis via an identical mechanism. Phospho-MAP kinase was markedly increased by all three estrogenic compounds (109 M) after 10 min of exposure (Fig.
2D).
SERMs reverse TGF-1-stimulated COL4A1 gene transcription.
COL4A1 gene transcription was increased in mesangial cells incubated
with TGF-
1 (2 ng/ml for 24 h; 137.7 ± 2.7, expressed as a
percentage of control values, P < 0.0001) (Fig.
3A). Physiological concentrations of all three estrogenic compounds (10
9 M)
reversed TGF-
1-stimulated COL4A1 gene transcription
(TGF-
1+estradiol: 96.0 ± 1.9, P < 0.0001 vs.
TGF-
1, P NS vs. control; estradiol alone: 107.9 ± 1.8, P NS vs. control; TGF-
1+LY-117018: 99.6 ± 2.7, P < 0.0001 vs. TGF-
1, P NS vs control;
LY-117018 alone: 105.6 ± 1.2, P NS vs. control;
TGF-
1+tamoxifen: 105.9 ± 0.7, P < 0.0001 vs.
TGF-
1, P NS vs. control; tamoxifen alone: 108.9 ± 1.2, P NS vs. control) (Fig. 3A).
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SERMs reverse ANG II-stimulated COL4A1 gene transcription.
ANG II (108 to 10
6 M) stimulated COL4A1
gene transcription in a dose-dependent manner, achieving statistical
significance at a concentration of 10
6 M (125.3 ± 2.1, expressed as a percentage of control values, P < 0.0001 vs. control). ANG II (10
6 M)-stimulated gene
transcription was reversed by a nonselective ANG II receptor antagonist
(ANG II antipeptide, 10
5 M; 100.3 ± 0.5, P NS vs. control, P < 0.00001 vs. ANG II;
ANG II antipeptide alone: 100.2 ± 0.6, P NS vs.
control) (Fig. 3B). A neutralizing antibody to TGF-
1
completely reversed the stimulatory effect of ANG II on COL4A1 gene
transcription: (100.4 ± 0.8, NS vs. control, P < 0.0001 vs. ANG II; anti-TGF-
1 antibody alone: 100.7 ± 0.5, P NS vs. control). IgG lacking anti-TGF-
1 specificity had
no effect on ANG II-stimulated COL4A1 gene transcription (126.4 ± 3.1, P NS vs. ANG II alone, P < 0.0001 vs.
control; IgG alone: 100.9 ± 0.4, P NS vs. control)
(Fig. 3B).
SERMs reverse ANG II-stimulated type IV collagen synthesis.
We next examined the effect of the estrogenic compounds on ANG
II-stimulated collagen IV protein synthesis as assessed by Western
blotting (Fig. 3D). ANG II (106 M) increased
type IV collagen synthesis (191.4 ± 18.9, P < 0.0001 vs. control, n = 7). ANG II-stimulated type IV
collagen synthesis was reversed by anti-TGF-
1 antibody (106.8 ± 8.9, P NS vs. control, P < 0.002 vs. ANG
II; anti-TGF-
1 antibody alone: 106.0 ± 6.6, P NS
vs. control). All three estrogenic compounds also completely reversed
the stimulatory effects of ANG II on collagen IV synthesis (ANG
II+estradiol: 100.1 ± 7.9, P NS vs. control,
P < 0.0001 vs. ANG II alone; estradiol alone:
102.3 ± 6.3, P NS vs. control; ANG II+LY-117018:
101.2 ± 5.8, P NS vs. control, P < 0.001 vs. ANG II alone; LY-117018 alone: 99.8 ± 3.8, P
NS vs. control; ANG II+tamoxifen: 109.8 ± 6.6, P NS
vs. control, P < 0.0001 vs. ANG II; tamoxifen alone:
106.7 ± 4.9, P NS vs. control) (Fig. 3D). The suppressive effects of the estrogenic compounds and anti-TGF-
1 antibody were not additive (ANG II+estradiol+anti-TGF-
1: 106.8 ± 8.9, P NS vs. ANG II+estradiol, P NS vs. ANG
II+anti-TGF-
1 antibody, P NS vs. control,
P < 0.002 vs. ANG II; ANG II+LY-117018+anti-TGF-
1 antibody: 102.2 ± 1.1, P NS vs. ANG II+LY-117018,
P NS vs. ANG II+anti-TGF-
1 antibody, P NS vs.
control, P < 0.0001 vs. ANG II; ANG
II+tamoxifen+anti-TGF-
1 antibody: 104.9 ± 0.9, P NS
vs. ANG II+tamoxifen, P NS vs. ANG II+anti-TGF-
1
antibody, P NS vs. control, P < 0.0001 vs.
ANG II) (Fig. 3D).
SERMs reverse ET-1-stimulated COL4A1 gene transcription.
ET-1 (109 to 10
6 M) stimulated COL4A1 gene
transcription in a dose-dependent manner, achieving statistical
significance at a concentration of 10
7 M (121.3 ± 1.3, P < 0.0001 vs. control). PD 0156707, an
ETA-selective receptor antagonist, completely reversed the
stimulatory effect of ET-1 (100.4 ± 2.9, P NS vs.
control, P < 0.0001 vs. ET-1; ETA receptor
antagonist alone: 100.1 ± 1.2, P NS vs. control) (Fig. 4A). A neutralizing antibody
to TGF-
1 also completely reversed the stimulatory effect of ET-1 on
COL4A1 gene transcription (99.1 ± 1.8, P NS vs.
control, P < 0.0001 vs. ET-1; anti-TGF-
1 antibody alone: 101.3 ± 1.0, P NS vs. control) (Fig.
4A). IgG lacking anti-TGF-
1 specificity had no effect on
ET-1-stimulated COL4A1 gene transcription (124.6 ± 2.6, P NS vs. ET-1 alone, P < 0.0001 vs.
control; IgG alone: 100.9 ± 0.4, P NS vs. control).
|
SERMs reverse ET-1-stimulated type IV collagen synthesis.
Last, we examined the effect of the estrogenic compounds on
ET-1-stimulated collagen IV protein synthesis as assessed by Western blotting (Fig. 4C). The ability of ET-1 to stimulate type IV
collagen synthesis was completely reversed by each of the following:
anti-TGF-1 neutralizing antibody, estradiol
(10
9 M), LY-117018 (10
9 M), and tamoxifen
(10
9 M). The suppressive effects of anti-TGF-
1
antibody were not additive to those of estradiol, LY-117018, or tamoxifen.
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DISCUSSION |
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SERMs are compounds that bind to the ER but have tissue-specific effects that may differ from those of estrogen itself (4, 12, 22). The promise of the SERMs lie in their ability to reproduce the beneficial effects of estrogen on bone, endothelium, and lipoprotein metabolism without reproducing the undesirable effects of estrogen on reproductive tissues (4, 12, 22). Raloxifene, a second-generation SERM, has a more favorable tissue-selectivity profile than does tamoxifen, a first-generation SERM (4, 12, 22). Although both compounds act as estrogen antagonists in mammary tissue, tamoxifen is a partial estrogen agonist in endometrial tissue whereas raloxifene is a complete estrogen antagonist in the uterus (4).
If these agents were to reproduce estradiol's suppressive effects on
mesangial cell collagen synthesis, they might prove clinically useful
in ameliorating progressive extracellular matrix accumulation and
resulting glomerulosclerosis in chronic renal disease without reproducing the adverse effects of estradiol on reproductive tissues. The potential clinical usefulness of second-generation SERMs in limiting progressive renal disease led us to explore in detail their
antifibrotic actions. In the present study we examined the effects of a
first-generation SERM (tamoxifen) and a second-generation SERM
(LY-117018) on the synthesis of type I and type IV collagen by cultured
mesangial cells. We also investigated the effects of the SERMs on the
ability of fibrogenic agents such as TGF-1, ANG II, and ET-1 to
stimulate mesangial cell COL4A1 gene transcription and type IV collagen synthesis.
We found that LY-117018 and tamoxifen suppress COL4A1 gene transcription and type IV collagen protein synthesis in a dose-dependent manner, with a potency identical to that of estradiol. Type I collagen synthesis was also suppressed by LY-117018 in a dose-dependent manner with a potency identical to that of estradiol but greater than that of tamoxifen.
We have previously shown that estradiol suppresses mesangial cell type I collagen synthesis by increasing the activity of AP-1 via upregulation of the mitogen-activated protein kinase pathway (25, 31). Our demonstration that the SERMs upregulate MAP kinase and increase the binding of nuclear extracts to an AP-1 binding site supports the hypothesis that the SERMs suppress type I collagen synthesis via a mechanism identical to what we described for estradiol.
We also examined the effect of the SERMs on the ability of the
fibrogenic agents TGF-1, ANG II, and ET-1 to stimulate COL4A1 gene
transcription and type IV collagen synthesis. We confirmed that the
stimulatory effects of ANG II and ET-1 on mesangial cell COL4A1 gene
transcription and type IV collagen synthesis are mediated by the
autocrine/paracrine effects of TGF-
1 (9,
10). We showed that estradiol, in physiological
concentrations, reverses the stimulatory effects of ANG II and ET-1 on
COL4A1 gene transcription and type IV collagen synthesis by
antagonizing the actions of TGF-
1.
LY-117018 and tamoxifen, in concentrations identical to the
physiological concentration of estradiol, reproduced the suppressive effects of estradiol on TGF-1-stimulated COL4A1 gene transcription and type IV collagen synthesis. In a manner identical to estradiol, the
SERMs also reversed the actions of ANG II and ET-1 on type IV collagen
via antagonism of the autocrine/paracrine effects of TGF-
1.
Estrogens, SERMs, and ICI-182780 (a pure ER antagonist) all bind to the ER but elicit different transcriptional responses depending on the specific cell type and gene promoter (22). The diversity of responses may arise due to interactions with 1) different ER isoforms (1, 28), 2) hormone response elements other than the estrogen response element (37), or 3) different gene- or cell-specific effector molecules (coactivators, corepressors, transcription factors, chromatin remodeling proteins) (1, 4, 12, 22). Differences in the conformation of complexes formed between ER and specific ligands may contribute to tissue- and cell type-specific genomic responses by facilitating or interfering with the ability of the ER to recruit cell-specific coactivators or corepressions or by interfering with the ability of the ER to interact with DNA response elements (4, 12, 22). In addition, ligand/tissue selectivity may be conferred by differential ligand activation of the constitutive hormone-independent transcription activation function domain of the ER (AF-1) vs. the hormone-inducible AF-2 domain (35). In this regard, we found no ligand/tissue selectivity with respect to the effects of estradiol, tamoxifen, and LY-117018 on the synthesis of type I and type IV collagen by murine mesangial cells.
In addition to the classic estrogen receptor (ER-), a second isotype
(ER-
) has recently been identified (15). Vascular smooth muscle and vascular endothelial cells from a variety of species
express both receptor subtypes (21). ER-
is the
predominant isotype expressed in human fetal kidney, with minor
expression of ER-
(3). Most studies have demonstrated
moderate expression of ER-
in the absence of ER-
in adult renal
tissue in humans, rats, and mice (14, 23,
34). However, other investigators have demonstrated ER
in rat kidneys (27). The varying ratios of ER-
and
ER-
in tissues that express both receptor isotypes may be involved
in the tissue- and cell type-specific effects of estrogens vs. SERMs
(29). ER-
and ER-
respond differently to ligands at
AP-1 sites (28). In the presence of ER-
, estrogen and
tamoxifen show agonist effects at AP-1 sites whereas raloxifene acts as
a partial agonist (28). In the presence of ER-
,
tamoxifen, raloxifene, and ICI-182780 show agonist activity at AP-1
sites, whereas estrogen acts as an antagonist of raloxifene
(28). In addition, at estrogen response elements, the
transcriptional activity of estrogen is greater when bound to ER-
than ER-
(11, 23). In cells that express
both receptor isotypes, heterodimer complexes may form that exhibit an
intermediate DNA binding affinity and transcriptional activity
(6, 29). In the present study we clearly
demonstrate the presence of both ER-
and ER-
in male murine
mesangial cells.
Genistein in micromolar concentrations inhibits tyrosine kinase
activity. In these concentrations, genistein stimulates type I
collagen synthesis in murine mesangial cells (25). In
contrast, genistein in nanomolar concentrations lacks tyrosine kinase
inhibitory activity but selectively binds to ER- (16,
20). We found that genistein in nanomolar concentrations
suppresses type I and type IV collagen synthesis by an ER-dependent
mechanism. These data suggest that ER-
mediates, at least in part,
the suppressive effects of estrogen on collagen synthesis. However, we
cannot exclude the possibility that functional redundancy exists
between ER-
and ER-
.
Because accumulation of glomerular extracellular matrix after renal injury is a precursor to the development of glomerular obsolescence and progressive loss of renal function, the ability of estrogens to inhibit fibrogenic cytokine-stimulated collagen IV synthesis may contribute to the protective effect of female gender on the progression of renal disease (33). Second-generation SERMs, such as LY-117018, may prove clinically useful in slowing the progression of chronic renal disease by reproducing the ability of estrogen to reduce glomerular matrix accumulation without reproducing its adverse effects on female reproductive tissues.
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ACKNOWLEDGEMENTS |
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This work was supported by a Grant-in-Aid from the American Heart Association, Heritage Affiliate.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Neugarten, Montefiore Medical Center, 111 E. 210 St., Bronx, NY 10467.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 1 November 1999; accepted in final form 6 April 2000.
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