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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2003-032154
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 247-255
Copyright © 2005 by The Endocrine Society

Cytochromes 1A1/1B1- and Catechol-O-Methyltransferase-Derived Metabolites Mediate Estradiol-Induced Antimitogenesis in Human Cardiac Fibroblast

Raghvendra K. Dubey, Edwin K. Jackson, Delbert G. Gillespie, Marinella Rosselli, Federica Barchiesi, Andree Krust, Hansjoerg Keller, Lefteris C. Zacharia and Bruno Imthurn

Department of Obstetrics and Gynecology, Clinic for Endocrinology, University Hospital Zurich (R.K.D., M.R., F.B., B.I.), 8091 Zurich, Switzerland; Center for Clinical Pharmacology, Departments of Medicine (R.K.D., E.K.J., D.G.G., L.C.Z., B.I.) and Pharmacology (E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213-2582; Arthritis and Bone Metabolism Therapeutic Division, Novartis Pharma Research (H.K.), Basel CH-40002, Switzerland; and Institut de Gènètique et de Biologie Molèculaire et Cellulaire (A.K.), 67404 Illkirch, France

Address all correspondence and requests for reprints to: Dr. Raghvendra K. Dubey, Department of Obstetrics and Gynecology, Clinic for Endocrinology, D217, NORD-1, Frauenklinik, University Hospital Zurich, CH-8091 Zurich, Switzerland. E-mail: raghvendra.dubey{at}usz.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) in the growth inhibitory effects of estradiol in cardiac fibroblasts (CFs) expressing functional estrogen receptors. 3-Methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and ß-naphthoflavone (CYP1A1/1A2 inducer) augmented, and 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor) blocked, the inhibitory effects of estradiol on serum-induced CF growth (DNA synthesis, cell number, and collagen synthesis). Neither ketoconazole (3A4 inhibitor) nor furafylline (selective 1A2 inhibitor) altered the antimitogenic effects of estradiol on CF growth. In contrast, ellipticine (selective 1A1 inhibitor), pyrene (selective 1B1 inhibitor), and {alpha}-naphthoflavone (1A1>1A2 inhibitor) abrogated the antimitogenic effects of estradiol on CF growth. OR486 (COMT inhibitor) also blocked the antimitogenic effects of estradiol in both the presence and absence of the CYP450 inducers. ICI182780 (estrogen receptor antagonist) attenuated the growth inhibitory effects of estradiol, but only at concentrations that inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). CFs expressed CYP1A1 and CYP1B1, isozymes that convert estradiol to hydroxyestradiols. Moreover, CFs metabolized estradiol to hydroxyestradiol, and 2-hydroxyestradiol to 2-methoxyestradiol. OR486 and quercetin (COMT inhibitor) blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol in CFs. We conclude that the antimitogenic effects of estradiol on CF growth are mediated in part by conversion to hydroxyestradiols via CYP1A1 and CYP1B1, followed by metabolism of hydroxyestradiols to methoxyestradiols by COMT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIAC FIBROBLASTS (CFs) comprise 60% of the total heart cells and contribute to pathological structural changes in the heart by undergoing proliferation, depositing extracellular matrix proteins, and replacing myocytes with fibrotic scar tissue (1, 2, 3). Consequently, abnormal growth of CFs may be importantly involved in the pathophysiology of cardiac remodeling induced by hypertension and myocardial infarction and may contribute to diastolic and systolic left ventricular dysfunction (1, 2, 3). Therefore, the development of drugs that modulate the growth of CFs may benefit patients with hypertension, myocardial infarction, or congestive heart failure.

Antimitogenic effects of estradiol on smooth muscle cell (SMC) growth plays a key role in mediating the antivasoocclusive effects of estradiol (4, 5, 6, 7). Like SMCs (8, 9, 10), CFs possess functional estrogen receptors (ERs) (11, 12) {alpha} and ß, and estradiol inhibits mitogen-induced growth in both SMCs (13, 14, 15, 16) and CFs (17, 18). Based on the conventional mechanisms of steroid action, the growth inhibitory effects of estradiol are thought to be mediated via ER{alpha} and/or ERß, expressed by cardiovascular cells (4, 7). However, recent findings that exogenous estradiol inhibits injury-induced SMC proliferation in mice lacking ER{alpha} (19), ERß (20), or both ER{alpha} and ERß (double knockout) (21) challenge this concept. Because the ER knockout models used in the above studies were not complete knockouts for ER{alpha}, the inhibitory effects of estradiol may have been due to residual ER activity. Indeed, in complete ER{alpha} knockout mice, developed in Strasbourg by Chambon and colleagues (22), the inhibitory effect of estradiol on injury-induced lesions was abolished; however, the lack of neointima formation in the ER{alpha} knockout compared with wild-type mice makes the data difficult to interpret (22). Also, estradiol failed to inhibit mitogen-induced growth of SMCs cultured from catechol-O-methyltransferase (COMT) knockout mice that expressed both ER{alpha} and ERß (23). Thus, whether the antimitogenic effects of estradiol are ER dependent or ER independent remains an open question.

Our previous studies demonstrate that estradiol, hydroxyestradiols, and methoxyestradiols potently inhibit rat CF growth (18). In this regard, hydroxyestradiols and methoxyestradiols, which are endogenous metabolites of estradiol with little or no affinity for ERs, are more potent inhibitors of CF growth than is estradiol, suggesting that the growth inhibitory effects of estradiol may actually be mediated by its downstream metabolites (18). If this hypothesis is correct, metabolites of estradiol, rather than estradiol per se, may be more effective in preventing cardiac remodeling and would be prime candidates for drug development.

The purpose of the present study was to extend our investigation of the role of estradiol metabolites in mediating the antigrowth effects of estradiol in human CFs. As depicted in Fig. 1Go, the metabolism of estradiol to catecholestradiols and methoxyestradiols is catalyzed by the sequential actions of cytochrome P450s (CYP450s) (24) and COMT (7, 24), which are expressed in the heart (25, 26). Therefore, to test our hypothesis, we studied the inhibitory effects of estradiol on DNA synthesis, collagen synthesis, and proliferation of human CFs in the presence and absence of modulators (selective inhibitors or activators) of CYP450 isozymes and COMT (Table 1Go and Fig. 1Go).



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  		FIG. 1. Schematic representation of the hypothesis and the experimental approach to demonstrate that in addition to ERs, the metabolism of estradiol to methoxyestradiols is an alternative pathway responsible for mediating the antimitogenic effects of 17ß-estradiol on CF growth. CYP1A2, CYP1A1, CYP1B1, CYP3A4, COMT, ABT (broad-spectrum CYP450 inhibitor; 10 µmol/liter), furafylline (FUR; selective CYP1A2 inhibitor; 10 µmol/liter), ellipticine (ELP; selective CYP1A1 inhibitor; 10 µmol/liter), {alpha}-naphthoflavone ({alpha}NA; selective CYP1A1 inhibitor; 10 µmol/liter), pyrene (PYR; selective CYP1B1 inhibitor; 5 nmol/liter), ketoconazole (KET; selective CYP3A4 inhibitor; 10 µmol/liter), phenobarbital (PB; broad-spectrum CYP450 inducer, 10 µmol/liter), 3-methylcholantherene (3-MC; broad spectrum CYP450 inducer; 10 µmol/liter), OR486 (COMT inhibitor; 10 µmol/liter), quercetin (QUER; COMT inhibitor; 10 µmol/liter), and ICI182780 [ICI*; inhibits CYP1A1/CYP1A2 and CYP1B1 isozymes of CYP450 at high (≥10 µmol /liter) concentrations] were studied. +, Inducer; –, inhibitor.

 

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  		TABLE 1. Specificities of the various compounds used to modulate (induce or inhibit) the metabolism of estradiol by various CYP450 isozymes and COMT in cardiac fibroblasts

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CF cultures and growth studies

Human female left ventricular CFs between second passage (Cell Applications, Inc., San Diego, CA) were cultured under standard tissue culture conditions in DMEM/Ham’s F-12 culture medium (Invitrogen Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% fetal calf serum (HyClone, Inc., Logan, UT) as described previously (27). Cells were cultured in steroid-free and phenol red-free medium. CFs between the third and fourth passages were used in all studies. The CF purity of greater than 97% was established by positive immunostaining with antibodies to vimentin and negative staining for sarcomeric actin (striated muscle), and von Willebrand factor VIII as described in detail previously (27).

Subconfluent CFs were growth-arrested for 48 h in the presence or absence of 10 µmol/liter of the broad-spectrum CYP450 inducers 3-methylcholantherene (3-MC), phenobarbital, or the CYP1A1/1A2 inducer ß-naphthoflavone. For [3H]thymidine incorporation, growth was initiated by treating growth-arrested cells for 20 h with DMEM supplemented with steroid-free fetal calf serum (FCS; 2.5%) containing or lacking fresh 3-MC, phenobarbital, or ß-naphthoflavone in the presence or absence of various treatments or vehicle. After 20 h of incubation, treatments were repeated with freshly prepared solutions, but were supplemented with [3H]thymidine (1 µCi/ml) for an additional 4 h. Incorporation of [3H]thymidine in the acid-insoluble fraction was subsequently measured on a scintillation counter using our previously described method (10).

To measure cell number, CFs were plated (5 x 103 cells/well of a 24-well tissue culture plate) and allowed to attach overnight. Cells were growth-arrested for 48 h and subsequently treated every 24 h for 4 d. On d 5, cells were dislodged by trypsinization and counted on a Coulter counter (Beckman Coulter, Fullerton, CA).

For [3H]proline incorporation, confluent monolayers of CFs were growth-arrested for 48 h in the presence or absence of 10 µmol/liter 3-MC, phenobarbital, or ß-naphthoflavone. Collagen synthesis was stimulated by treating cells for 48 h with 2.5% FCS in the presence of L-[3H]proline (1 µCi/ml) and 3-MC, phenobarbital, or ß-naphthoflavone with various other treatments. Incorporation of [3H]proline in the acid-insoluble fraction was subsequently measured on a scintillation counter using our previously described method (10). Moreover, confluent monolayers of CFs were used to preclude the influence of changes in cell number.

To assess whether 2-methoxyestradiol inhibits growth of CFs in the absence of ERs, we evaluated and compared its antimitogenic effects on CFs cultured from the left ventricles of female mice completely lacking both ER{alpha} and ERß (ER-KO) and their wild-type (WT) litter mates (Strasbourg mice, Institut de Gènètique et de Biologie Molèculaire et Cellulaire, Illkirch, France). Briefly, CFs were isolated from the left ventricles using the enzymatic dispersion and were cultured under standard tissue culture conditions in DMEM/Ham’s F-12 plus 10% steroid-free FCS as described previously (27). A purity of greater than 98% was established by positive immunostaining with antibodies to vimentin and negative staining for sarcomeric actin (striated muscle) and von Willebrand factor VIII as described in detail previously (27). To assess the antimitogenic effects of 2-methoxyestradiol, CFs cultured from ER-KO and WT mice were plated in 24-well plates, growth-arrested for 48 h by feeding DMEM containing 0.4% albumin, and subsequently stimulated with 2.5% FCS in the presence and absence of 1–100 nmol/liter 2-methoxyestradiol. Cell growth was assessed by counting cells on d 6 (treatments changed every 48 h) or DNA synthesis after 24 h of treatment, as described above.

To ensure that the various treatments did not adversely influence cell viability, trypan blue exclusion and 3-[4,5-dimethylthiozol-2-yl] diphenyl tetrazolium bromide (MTT) assays were conducted in cells treated in parallel. For the trypan blue exclusion assay, cells were incubated for 5 min with 0.4% trypan blue solution in Hanks’ balanced salt solution, and subsequently cells taking up the dye were counted microscopically. For the MTT assay, we used a modified colorimetric assay based on the selective ability of living cells to reduce the yellow dye, MTT.

Metabolism studies

To assess whether CFs metabolize catecholestradiols to methoxyestradiols, confluent monolayers of CFs were incubated with 2-hydroxyestradiol for 4 h, internal standard (16{alpha}-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 HPLC with UV detection using gradient elution (28).

To investigate whether CFs metabolize estradiol to 2/4-hydroxyestradiol, we used 2,4–17ß-[3H]estradiol, which upon hydroxylation to 2/4-hydroxyestradiol releases [3H]H2O in a stoichiometric fashion (29). Measuring radiolabeled [3H]H2O formation is well established to provide a reliable estimate for the conversion of estradiol to 2-/4-hydroxyestradiol (29). Briefly, CFs grown to 70–80% confluence in six-well tissue culture dishes and pretreated for 48 h with 10 µmol/liter phenobarbital were fed 2 ml DMEM/Ham’s F-12 and supplemented with 2 µCi/ml [3H]2,4-ß-estradiol (specific activity, 29 Ci/mmol; Sigma-Aldrich Corp., St. Louis, MO) in the presence and absence of 1–50 µmol/liter ICI182780. After 20 h, the supernatants were collected, activated charcoal was added to each supernatant (final concentration, 1 mg/ml), and the samples were incubated overnight with gentle shaking at 4 C. Subsequently, 100 µl protamine sulfate (10 mg/ml) were added to the supernatants, and the samples were centrifuged at 3000 x g at 4 C. Next, 1 ml supernatant was collected, and the amount of [3H]H2O formed was assayed by counting on a ß-scintillation counter. 2,4-[3H]Estradiol incubated in absence of cells and processed in parallel served as the control and for subtracting the background counts after extraction.

CYP1A1, CYP1B1, ER{alpha}, and ERß expression studies

To investigate whether CFs express CYP1A1 and CYP1B1, cell lysates from cultured CFs were analyzed by Western blotting and probed with antibodies to CYP1A1 (rabbit antihuman polyclonal antibodies; Chemicon International, Inc., Temecula, CA) and CYP1B1 (rabbit antihuman polyclonal antibodies; Gentest Corp., Woburn, MA) as described previously (28). To investigate the expression of ER{alpha} and ERß, CFs grown to subconfluence were lysed and analyzed by Western blotting using specific antibodies against ER{alpha} (Alexis, 210-201-C050, no cross-reactivity with ERß) or ERß (Alexis, 210-180-C050, no cross-reactivity with ER{alpha}) as previously described in detail (30).

ER activation assays

To investigate whether ER activation occurs in CFs under the culture conditions used and whether ICI182780 blocks estrogen-induced activation, we conducted estrogen response element (ERE)-luciferase reporter assays in CFs overexpressing ER{alpha}. Briefly, cells were cultured in phenol red-free DMEM/Ham’s F-12 supplemented with 10% delipidated FCS, 1% nonessential amino acids, and antibiotics. For transfection, 80,000 cells were seeded in 24-well plates in 500 µl medium. They were transfected the next day with ERE reporter plasmid (ERE2.TK.Luc), ER{alpha} expression plasmid (pcDNA3.1.hERa), or empty vector (pcDNA3.1) and cytomegalovirus-ß-galactosidase expression plasmid as an internal control using SuperFect (Qiagen, Basel, Switzerland). After 3 h, the transfection medium was replaced by fresh medium containing 17ß-estradiol (10 nmol/liter), ICI182780 (100 nmol/liter), 17ß-estradiol (10 nmol/liter) plus ICI18270(100 nmol/liter), or solvent controls. Luciferase and ß-galactosidase activities were measured 20 h later in cell extracts using passive lysis buffer extraction buffer, luciferase, and the ß-galactosidase enzyme assay system (Promega Corp., Wallisellen, Switzerland).

To assess whether ERs expressed in cultured CFs were functional, we used a sensitive ELISA-based ER transcription factor assay kit (TransAM ER kit, Active Motif, Carlsbad, CA). Briefly, nuclear extracts of CFs treated for 20 h with or without 17ß-estradiol (10 nmol/liter), ICI182780 (100 nmol/liter), or 17ß-estradiol (10 nmol/liter) plus ICI18270(100 nmol/liter) were isolated and analyzed according to the manufacturer’s specifications.

Statistics

Statistical significant (P < 0.05) was assessed by ANOVA, Student’s t test, or Fisher’s least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In growth-arrested CFs, treatment with 2.5% FCS induced DNA synthesis ([3H]thymidine incorporation), collagen synthesis ([3H]proline incorporation), and proliferation (cell number) by 7.6 ± 0.9, 6.4 ± 1.3, and 9.2 ± 1.7-fold, respectively. Estradiol and its metabolites (2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol) inhibited FCS-induced DNA synthesis (Fig. 2AGo), collagen synthesis (Fig. 2BGo) and proliferation (Fig. 2CGo). The relative potencies for inhibition of FCS-induced growth were 2methoxyestradiol > 4-methoxyestradiol ≥ 2-hydroxyestradiol ≥ 4-hydroxyestradiol > estradiol.



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  		FIG. 2. Effects of increasing concentrations of estradiol (ßE), 2-hydroxyestradiol (2OHE), 4-hydroxyestradiol (4OHE), 2-methoxyestradiol (2ME), and 4-methoxyestradiol (4ME) on FCS-induced [3H]thymidine incorporation (A), collagen synthesis (B), and cell number (C) in human cardiac fibroblasts. The results are presented as the percent change from control (CFs treated with FCS alone). Values for each data point represent the mean ± SEM from three separate experiments conducted in quadruplicate. *, P < 0.05 vs. control treated with FCS alone; §, significantly (P < 0.05) different from estradiol alone. Symbols defining the treatments apply for all panels.

 
Incubation of CFs for 48 h with CYP450 inducers, 3-MC (10 µmol/liter) (31), phenobarbital (10 µmol/liter) (31), or ß-naphtoflavone (10 µmol/liter) (32) and with a CYP450 inhibitor [1-aminobenzotriazole (ABT); 0.1–10 µmol/liter] (28, 33) did not affect FCS-induced DNA synthesis, cell proliferation, or collagen synthesis. In contrast, the effects of estradiol (1–100 nmol/liter) on cell growth were augmented significantly by 3-MC, phenobarbital (CYP450 inducers), and ß-naphthoflavone (selective CYP1A1/1A2 inducer). In this regard, the ability of estradiol (1 nmol/liter) to inhibit FCS-induced proliferation of CFs on d 5 of the growth curve was increased from 19% to 47%, 52%, and 36% by the CYP450 inducers 3-MC, phenobarbital, and ß-naphthoflavone, respectively (Fig. 3Go). In contrast, ABT, a broad-spectrum CYP450 inhibitor, decreased the inhibitory effects of estradiol on cell proliferation, DNA synthesis, and collagen synthesis (Fig. 4Go).



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  		FIG. 3. Modulatory effects of 10 µmol/liter CYP450 inducers (3MC, 3-methylcholantherene; PB, phenobarbital; ßNA, ß-naphthoflavone) on the inhibitory effect of estradiol (ßE) on 2.5% FCS-induced DNA synthesis (A), collagen synthesis (B), and proliferation (concentration of ßE of 1 nmol/liter; C) of CFs. Values for each data point represent the mean ± SEM from three separate experiments conducted in quadruplicate. *, P < 0.05 vs. cells treated with FCS alone; §, significantly (P < 0.05) different from estradiol alone. Symbols defining the treatments in A apply for B also.

 


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  		FIG. 4. Effects of broad-spectrum and specific inhibitors of CYP450 isozymes on the inhibitory actions of 1–100 nmol/liter estradiol. The inhibitory effect of estradiol (1–100 nmol/liter) on 2.5% FCS-induced DNA synthesis (A), collagen synthesis (B), and CF proliferation (concentration of ßE, 100 nmol/liter; C) were determined in the presence or absence of pyrene (PYR; selective CYP1B1 inhibitor; 5 nmol/liter), {alpha}-naphthoflavone ({alpha}NA; selective CYP1A1 inhibitor; 10 µmol/liter), ellipticine (ELP; selective CYP1A1 inhibitor; 10 µmol/liter), furafylline (FUR; selective CYP1A2 inhibitor; 10 µmol/liter), ketoconazole (KET; selective CYP3A4 inhibitor; 10 µmol/liter), or ABT (broad-spectrum CYP450 inhibitor; 10 µmol/liter). Values for each data point represent the mean ± SEM from three separate experiments conducted in quadruplicate. *, P < 0.05 vs. cells treated with FCS alone; §, significantly (P < 0.05) different from estradiol alone. Symbols defining the treatments in A and B, apply to both A and B.

 
Ellipticine (selective CYP1A1 inhibitor; 10 µmol/liter) (34), pyrene (selective CYP1B1 inhibitor; 5 nmol/liter) (35), and {alpha}-naphthoflavone (selective CYP1A1 inhibitor; 10 µmol/liter) (35), but not ketoconazole (selective CYP3A4 inhibitor; 10 µmol/liter) (36) or furafylline (selective CYP1A2 inhibitor; 10 µmol/liter) (37) attenuated the growth inhibitory effects of estradiol on DNA synthesis, cell proliferation, and collagen synthesis (Fig. 4Go). On d 5 of the growth study, ellipticine, pyrene, and {alpha}-naphthoflavone reduced the inhibitory effect of 1 nmol/liter estradiol on cell proliferation from 29% to 6%, 3%, and 14%, respectively. The ability of ellipticine and pyrene to attenuate the inhibitory effect of estradiol (100 nmol/liter) on DNA synthesis, cell number, and collagen synthesis was concentration dependent (Fig. 5Go, A and B). In this regard, pyrene was more potent than ellipticine in attenuating the inhibitory effect of estradiol on CF growth.



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  		FIG. 5. A, Concentration-dependent effects of OR486 (COMT inhibitor), quercetin (QUE; COMT inhibitor), ICI182780 (ICI; ER antagonist), ABT (broad-spectrum CYP450 inhibitor), ellipticine (ELP; selective CYP1A1 inhibitor), and pyrene (selective CYP1B1 inhibitor) on the inhibitory effect of estradiol (ßE; 100 nmol/liter) on FCS-induced DNA synthesis. *, Significant reversal of the inhibitory effects of estradiol. B, Concentration-dependent effects of ellipticine and pyrene on the inhibitory effect of estradiol (ßE; 100 nmol/liter) on FCS-induced collagen synthesis and cell number. *, Significant reversal of the inhibitory effect of estradiol. C, Antagonistic effects of concentrations of ICI182780 (ICI) that inhibit estradiol (ßE) metabolism (50 µmol/liter) and do not inhibit estradiol metabolism (1 µmol/liter) on the inhibitory effects of 1 and 50 nmol/liter estradiol, respectively, on FCS-induced proliferation of SMCs. The ratio of estradiol and ICI182780 was 1:1000 under both treatment conditions. The data are presented as a percentage of the control, where 100% is defined as the increase in cell number in response to FCS alone. Values are the mean ± SEM from four separate cultures. *, P < 0.05 vs. SMCs treated with FCS alone; §, significant (P < 0.05) reversal of the inhibitory effects of estradiol.

 
The ER antagonist, ICI182780 (38), attenuated the inhibitory effect of estradiol on DNA synthesis in a concentration-dependent fashion (Fig. 5AGo). However, ABT, ellipticine, pyrene, quercetin, and OR486 were more potent than ICI182780 in antagonizing the inhibitory effect of estradiol on CF growth (Fig. 5AGo). The lowest concentration of ICI182780 that significantly reduced the inhibitory effects of 100 nmol/liter estradiol was 10 µmol/liter. At a concentration of 50 µmol/liter, ICI182780 completely blocked the inhibitory effects of 100 nmol/liter estradiol (Fig. 5AGo). Although 1 µmol/liter ICI182780 was unable to block the inhibitory effects of 1 nmol/liter estradiol, 50 µmol/liter ICI182780 was able to block the inhibitory effects of 50 nmol/liter estradiol, even though the estradiol to ICI182780 ratio was 1:1000 in both cases (Fig. 5CGo). These findings suggest that ICI182780 abrogates the growth inhibitory effects of estradiol at high (≥10 µmol/liter), but not low (<10 µmol/liter), concentrations.

The COMT inhibitors, quercetin and OR486 (28, 39), also blocked the inhibitory effect of estradiol on DNA synthesis (Fig. 6AGo), cell proliferation (Fig. 6BGo), and collagen synthesis (Fig. 6CGo). The inhibitory effect of 2-hydroxyestradiol and 4-hydroxyestradiol, but not 2-methoxyestradiol and 4-methoxyestradiol, on CF proliferation, DNA synthesis, and collagen synthesis (Fig. 7Go) were blocked by quercetin and OR486. In contrast to quercetin and OR486, high concentrations (50 µmol/liter) of ICI182780 did not block the growth inhibitory effect of either 2- and 4-hydroxyestradiol or 2- and 4-methoxyestradiol (Fig. 7Go).



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  		FIG. 6. A, B, and C show the effects of OR486 (10 µmol/liter; COMT inhibitor) and quercetin (QUE; 10 µmol/liter; COMT inhibitor) on the inhibitory effects of estradiol (ßE) on FCS-induced DNA synthesis (A), cell number (B), and cell proliferation (cell number; C), respectively. Values for each data point represent the mean ± SEM from three separate experiments conducted in quadruplicate. *, P < 0.05 vs. cells treated with FCS alone or control; §, significantly (P < 0.05) different from estradiol alone.

 


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  		FIG. 7. Inhibitory effects of 0.1 µmol/liter 2-hydroxyestradiol (2-OE), 4-hydroxyestradiol (4-OE), 2-methoxyestradiol (2-ME), and 4-methoxyestradiol (4-ME) on 2.5% FCS-induced DNA synthesis, collagen synthesis, and cell number of CFs in the presence and absence of the COMT inhibitors quercetin (QUE; 10 µmol/liter) and OR486 (10 µmol/liter) and the ER antagonist ICI182780 (ICI; 50 µmol/liter), Values are the mean ± SEM from three separate experiments conducted in quadruplicate. §, P < 0.05 vs. control; *, significant reversal of inhibitory effect.

 
CFs converted 2-hydroxyestradiol to 2-methoxyestradiol, and this was blocked by quercetin and OR486, but not by a high concentration (10 µmol/liter) of ICI182780 (Fig. 8AGo). Moreover, CFs expressed both CYP1A1 and CYP1B1 protein (Fig. 8BGo), and under basal conditions the expression of CYP1B1 was greater than CYP1A1 (Fig. 8BGo). In CFs treated with 3-MC and phenobarbital, the expression of CYP1A1 and CYP1B1 isozymes was increased. Additionally, incubation of CFs with 2,4–17ß-[3H]estradiol resulted in hydroxyestradiol formation, as assayed by the release of [3H]H2O. The formation of [3H]H2O, a stoichiometric indicator for hydroxyestradiol formation, was inhibited by the CYP450 inhibitor ABT (10 µmol/liter) as well as by high (≥10 µmol/liter), but not low (<10 µmol/liter), concentrations of the ER antagonist ICI182780 (Fig. 8CGo). Trypan blue exclusion tests and MTT assay indicated no loss in the viability of cells treated with various agents.



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  		FIG. 8. A, The capability of CFs to metabolize 2-hydroxyestradiol to 2-methoxyestradiol, and the inhibitory effects of quercetin (QUE; 10 µmol/liter), OR486 (OR; 10 µmol/liter), and ICI182780 (ICI; 10 µmol/liter) on the metabolism of 2-hydroxyestradiol (5 µmol/liter) to 2-methoxyestradiol (2-ME) by CFs. Values for each data point represent the mean ± SEM from three separate experiments conducted in quadruplicate. *, P < 0.05 vs. control (CON; cells treated with 2-OH-E alone), significant inhibition of 2-methoxyestradiol formation. B, Representative Western blot depicting the expression of CYP1A1 and CYP1B1 in CFs treated with or without 10 µmol/liter 3-methylcholantherene (3-MC) or phenobarbital (PB). C, Graph showing the metabolic capability of CFs to convert estradiol to hydroxyestradiol using the radiolabeled 2'4'-ß-[3H]estradiol assay for determining the stoichiometric conversion of estradiol to hydroxyestradiol (OH-E) by analyzing [3H]H2O release. The formation of [3H]H2O was assessed in CFs treated for 20 h with medium (control), phenobarbital (PB; 10 µmol/liter), or PB plus ABT (10 µmol/liter). Moreover, the line graph depicts the concentration-dependent inhibitory effect of ICI182780 (ICI; 1–50 µmol/liter) on the release of [3H]H2O from [3H]estradiol after 2/4-hydroxylation by CFs. Values for each data point represent the mean ± SEM from four samples. *, P < 0.05 vs. controls (CFs in medium alone); §, P < 0.05, significant inhibition of [3H]H2O formation.

 
The CFs used in the experiments expressed both ER{alpha} and ERß (Fig. 9AGo). In CFs overexpressing ER{alpha}, ERE-luciferase reporter assays demonstrated that under the culture conditions used, a low concentration (100nmol/liter) of ICI182780 abrogated estradiol (10 nmol/liter)-induced ER{alpha} activation (Fig. 9BGo). Additionally, using an ELISA-based ER activation assay, ER activation was observed in normal CFs treated with 17ß-estradiol (10 nmol/liter), and this effect was blocked by a low concentration (100 nmol/liter) of ICI182780. The ER activation values (absorbance/OD at 450 nm) in CFs treated with medium, estradiol, ICI182780, and estradiol plus ICI182780 were 0.326 ± 0.04, 1.68 ± 0.32 (P < 0.05 vs. CFs in medium), 0.41 ± 0.02, and 0.64 ± 0.2 (P < 0.05 vs. CFs treated with estradiol), respectively. Treatment with 2-methoxyestradiol concentration-dependently inhibited both DNA synthesis and cell number induced by 2.5% FCS in CFs cultured from ER-KO and WT mice (Fig. 9Go, C and D). Moreover, the inhibitory actions of 2-methoxyestradiol were similar in CFs cultured from WT and ER-KO (Fig. 9Go, C and D).



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  		FIG. 9. A, Representative Western blot depicting the expression of ER{alpha} and ERß in the CFs used in our experiments. B, Bar graph showing the inhibitory effect of ICI182780 (ICI; 100 nmol/liter) on estradiol (ß-Est; 10 nmol/liter)-induced ER activation in CFs overexpressing ER{alpha}, using the ERE-reporter luciferase assay. Values represent the mean ± SEM. Similar effects were observed in normal CFs using the ELISA-based ER activation assay. C and D, Line graphs showing the concentration-dependent inhibitory effects of 2-methoxyestradiol on 2.5% FCS (steroid free)-induced DNA synthesis (C) and cell number (D) in CFs cultured from wild-type (WT) and ER{alpha} and ERß double-knockout mice (ERKO). Values for each data point represent the mean ± SEM from four samples. *, P < 0.05 vs. control (CFs treated with FCS alone).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we observed that 2- and 4-hydroxyestradiol and their methylated metabolites, 2- and 4-methoxyestradiol, are more potent than estradiol in reducing CF growth. We also found that the inhibitory effect of estradiol on CF growth is enhanced by CYP450 inducers and abolished by a broad-spectrum CYP450 inhibitor. Finally, we found that the inhibitory effect of estradiol on CF growth is blocked by selective inhibitors of CYP1A1 and CYP1B1, that CFs metabolize hydroxyestradiols to methoxyestradiols, and that CFs express CYP1A1 and CYP1B1 isozymes as well as metabolize estradiol to hydroxyestradiols and 2-hydroxyestradiol to 2-methoxyestradiol. These findings support the hypothesis that in CFs, sequential conversion of estradiol to methoxyestradiols by CYP1A1/CYP1B1 and COMT mediates in part the inhibitory effect of estradiol on CF growth.

In some tissue, CYP1A1 and CYP1B1 importantly contribute to the hydroxylation of estradiol (24, 28, 33). Our findings with ABT are consistent with the involvement of CYP1A1 and CYP1B1 in the conversion of estradiol to inhibitory metabolites in CFs. However, because ABT is a broad-spectrum CYP450 inhibitor, studies using ABT cannot determine the contributions of specific CYP450 isozymes. In contrast, our finding that the growth inhibitory effect of estradiol is attenuated by ellipticine and pyrene, selective inhibitors of CYP1A1 and CYP1B1, respectively (28, 35), provides strong evidence that the inhibitory effect of estradiol is mediated primarily by CYP1A1 and CYP1B1. This conclusion is strengthened by the observations that the inhibitory effect of estradiol is increased by exposure of CFs to ß-naphthoflavone, a selective inducer of CYP1A1 (32), and that CFs express both CYP1A1 and CYP1B1.

Estradiol can also be hydroxylated by CYP3A4 and CYP1A2 (7, 24, 33), and it is conceivable that these isozymes of CYP450 also mediate in part the conversion of estradiol to growth inhibitory metabolites. In this regard, our observation that the inhibitory effects of estradiol are not blocked by ketoconazole, a CYP3A4 inhibitor, nor by furafylline, a CYP1A2 inhibitor, indicates that these CYP450 isozymes do not participate in mediating the inhibitory effect of estradiol in CFs.

Seemingly at odds with our hypothesis, high concentrations (>10 µmol/liter) of the ER antagonist ICI182780 attenuate the inhibitory effect of estradiol on CF growth. However, the molecular structure of ICI182780 is similar to the structure of estradiol, and it is therefore possible that ICI182780 competes with estradiol for CYP450s and blocks the hydroxylation of estradiol. Indeed, in a previous study we found that in human lymphoblastoid cells expressing CYP1A1 and in supersomes expressing CYP1B1, ICI182780 inhibits the metabolism of estradiol to 2-hydroxyestradiol and 4-hydroxyestradiol (28, 40). Importantly, in the present study we found that blockade of estradiol-induced inhibition by ICI182780 is independent of the estradiol to ICI182780 ratio, but is dependent on whether the concentration of ICI182780 inhibits estradiol metabolism. Moreover, we observed that the metabolism of estradiol to hydroxyestradiols was inhibited by high concentrations (>10 µmol/liter) of ICI182780 that block the antimitogenic effects of estradiol, but not by low concentrations (<10 µmol/liter) of ICI182780 that do not block the antimitogenic effects of estradiol. These findings support the conclusion that ICI182780, at high concentrations (>10 µmol/liter), blocks the inhibitory effect of estradiol on CFs by preventing the metabolism of estradiol to catecholestradiols, the precursors of methoxyestradiols.

In the present study we also observed that the inhibitory effects of estradiol and hydroxyestradiols, but not methoxyestradiols, on CF growth are reduced by the COMT inhibitors quercetin and OR486 (28, 39), drugs that have no binding affinity for ERs (40, 41). In contrast, even a high concentration (50 µmol/liter) of ICI182780 does not block the inhibitory effects of hydroxyestradiols or methoxyestradiols on CF growth. This is strong evidence that the metabolism by COMT of hydroxyestradiols to methoxyestradiols mediates the inhibitory effect of hydroxyestradiols on CF growth. Also, these findings demonstrate that the inhibitory effects of hydroxyestradiols and methoxyestradiols are ER independent, as would be anticipated because of the low affinity of hydroxyestradiols and methoxyestradiols for ERs. The hypothesis that the inhibitory effect of estradiol is due to its conversion to methoxyestradiols is additionally supported by our observation that CFs metabolize 2-hydroxyestradiol to 2-methoxyestradiol and that this metabolic step is blocked by quercetin and OR486.

Our finding that methoxyestradiols inhibit the growth of CFs lacking both ER{alpha} and ERß suggests that methoxyestradiols inhibit CF growth via an ER-independent mechanism. Under the culture conditions used, human CFs express ER{alpha} and ERß, and estradiol-induced ER activation is blocked by a low concentration (100 nmol/liter) of ICI182780 in normal CFs. These results indicate that ERs are functionally active in our cellular system and imply that the antimitogenic effect of estradiol is ER independent and not due to the lack of functionally active ERs. The finding that ICI182780 blocks estradiol-induced ER activation in both normal CFs as well as in CFs overexpressing ER{alpha} suggests that the antagonistic actions of ICI182780 are not influenced by the culture conditions.

Although our findings provide evidence that the antimitogenic effects of estradiol are ER independent, multiple ER-dependent mechanisms may still play a prominent role in mediating specific protective actions of estradiol on the cardiovascular system. In this context, via ER-dependent mechanisms, estradiol induces the synthesis of cardioprotective molecules, such as nitric oxide (42), vascular endothelial growth factor (43), and prostacyclins (44), and induces endothelial cell growth (45). Moreover, the antimitogenic effects of estradiol via conversion to methoxyestradiols may be only mimicked by estrogens that are metabolized to methoxyestradiols, and an ER-dependent mechanism may still play an important role in mediating the growth regulatory effects of estrogens not metabolized to methoxyestradiols.

The conclusion that methoxyestradiols mediate the antigrowth effects of estradiol in CFs has important clinical implications. In postmenopausal women with an intact uterus, hormone replacement therapy with conjugated equine estrogens plus medroxyprogesterone not only does not benefit postmenopausal women, but, in fact, increases the risk of cancer, stroke, myocardial infarction, and thromboembolic disease (46, 47). Unlike estrogenic compounds, 2-methoxyestradiol decreases the growth of cancer cells (24), and, in fact, is being developed as an anticancer drug for breast and prostate cancers (48, 49). It is conceivable that 2-methoxyestradiol could be used clinically to prevent cardiac remodeling in women without increasing the risk of cancer, stroke, myocardial infarction, or thromboembolic disease. Because 2-methoxyestradiol is nonfeminizing (50), it could be of therapeutic benefit in men. Our findings may also provide leads to help explain the recent findings that significantly more adverse cardiovascular events were observed in postmenopausal women taking estrogen plus progestin than in those taking estrogen alone. Because progesterone inhibits the conversion of estradiol to hydroxyestradiol (51) and blocks the antimitogenic effects of estradiol (52), it is possible that progestins may abrogate the antimitogenic actions of estradiol by inhibiting the formation of hydroxyestradiols; this possibility needs to be further investigated.

Finally, our findings imply that local vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of circulating estradiol. Thus, interindividual differences, either genetic or acquired, in the cardiovascular metabolism of estradiol may define a given female’s risk of cardiovascular disease and influence the cardiovascular benefit she receives from estrogen replacement therapy in the postmenopausal state.

In conclusion, the findings of this study support the concept that estradiol inhibits the activity of CFs via the local metabolism to methoxyestradiols by CYP1A1/CYP1B1 and COMT. Our results suggest that methoxyestradiols may attenuate cardiac remodeling without causing estrogenic adverse effects.

Study limitations

Our findings provide evidence that the antimitogenic effects of estradiol are mediated via an ER-independent mechanism that involves its local conversion to methoxyestradiol. However, these conclusions are largely derived by using pharmacological agents, inducers, and inhibitors of enzymes involved in the sequential metabolism of estradiol and are known to exert nonspecific effects on proteins that were not the focus of the current investigation. Although treatment with the agents alone did not influence FCS-induced growth, future studies using molecular tools to suppress, delete, or induce CYP450 or COMT are required to confirm our findings.


   Footnotes
 
This work was supported by Swiss National Science Foundation Grants 32-64040.00 and 3200BO-106098/1, and NIH Grant HL-69846.

First Published Online October 26, 2004

Abbreviations: ABT, 1-Aminobenzotriazole; CF, cardiac fibroblast; COMT, catechol-O-methyltransferase; CYP450, cytochrome P450; ER, estrogen receptor; ERE, estrogen response element; ER-KO, ER knockout; FCS, fetal calf serum; 3-MC, 3-methylcholantherene; MTT, 3-[4,5-dimethylthiozol-2-yl] diphenyl tetrazolium bromide; SMC, smooth muscle cell; WT, wild type.

Received November 30, 2003.

Accepted September 28, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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