Methoxyestradiols Mediate the Antimitogenic Effects of 17β-Estradiol
Direct Evidence From Catechol-O-Methyltransferase–Knockout Mice
Background— Studies using pharmacological agents suggest but do not prove that the antimitogenic effects of estradiol are caused by conversion of estradiol to hydroxyestradiols (mediated by CYP450s) followed by methylation of hydroxyestradiols to methoxyestradiols (mediated by catechol-O-methyltransferase, COMT).
Methods and Results— To test this hypothesis more rigorously, we used aortic smooth muscle cells (SMCs) from mice lacking COMT (COMT-KO). Wild-type (WT) but not COMT-KO SMCs efficiently converted 2-hydroxyestradiol to 2-methoxyestradiol. Both WT and COMT-KO SMCs expressed estrogen receptors. Estradiol and 2-hydroxyestradiol concentration-dependently inhibited serum-induced DNA synthesis, cell numbers, and collagen synthesis in WT but not COMT-KO SMCs. 2-Methoxyestradiol inhibited DNA synthesis, cell numbers, and collagen synthesis in both WT and COMT-KO SMCs.
Conclusions— These data provide strong evidence that the vascular antimitogenic effects of estradiol are estrogen receptor–independent and involve the sequential conversion of estradiol to hydroxyestradiols and then to methoxyestradiols.
Received September 29, 2002; revision received October 27, 2003; accepted October 30, 2003.
Seventeen β-estradiol (estradiol) inhibits the growth of vascular smooth muscle cells (SMCs), even in mice lacking estrogen receptors (ERs).1–3 Because methoxyestradiols (major endogenous metabolites of estradiol with no affinity for ERs) inhibit growth of cancer cells,4 it is feasible that the vascular antimitogenic effects of estradiol are mediated by methoxyestradiols. Using pharmacological agents, previous studies demonstrate that metabolism of estradiol to hydroxyestradiols by CYP450 followed by methylation of hydroxyestradiols by catechol-O-methyltransferase (COMT) to form methoxyestradiols is critical for estradiol-induced inhibition of SMC growth.5 In the present study, we use aortic SMCs from COMT-knockout (COMT-KO) mice6 that express ERs but lack methylation capability to test our hypothesis that the vascular antimitogenic effects of estradiol are mediated via an ER-independent mechanism involving formation of methoxyestradiols.
COMT-KO mice were developed by Dr Karayiorgou.6 Aortic SMCs were cultured from male mice by the explant technique. SMC purity was tested by immunostaining with SMC-specific antibodies as described previously.7 Cells in passage 2 were used. [3H]Thymidine incorporation, [3H]proline incorporation, and cell proliferation were conducted as previously.7 The absence of COMT was confirmed by incubating SMCs from wild-type (WT) and COMT-KO mice for 2 hours with 2-hydroxyestradiol (2 μmol/L) and analyzing 2-hydroxyestradiol/2-methoxyestradiol by high-performance liquid chromatography, as previously.8 ERα and ERβ expression were analyzed by Western blots.9 Experiments were conducted in triplicate (repeated 3 or 4 times). Results are expressed as mean±SEM. Statistical analyses were performed with ANOVA and Fisher’s least significant difference test. A value of P<0.05 was considered statistically significant.
WT and COMT-KO SMCs were morphologically similar. SMCs stained positive for SMC-specific actin (Figure 1A) and negative for von Willebrand factor VIII or vimentin. Both WT and COMT-KO SMCs had similar growth responses to FCS (0.25% to 10%; Figure 1B). WT but not COMT-KO SMCs converted 2-hydroxyestradiol to 2-methoxyestradiol (Figure 1C). Methylation activity was observed in WT but not COMT-KO liver homogenates incubated with 2-hydroxyestradiol. Both WT and COMT-KO SMCs expressed ERα/β (Figure 1D).
FCS (2.5%) stimulated [3H]thymidine and [3H]proline incorporation by 6- to 8-fold (P<0.001). In WT SMCs, 1 to 100 nmol/L of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol inhibited [3H]thymidine and [3H]proline incorporation in a concentration-dependent manner (Figure 1, E and F) in the following order of potency: 2-methoxyestradiol >2-hydroxyestradiol >estradiol. In COMT-KO SMCs, estradiol and 2-hydroxyestradiol had no inhibitory effects on FCS-induced [3H]thymidine or [3H]proline incorporation, whereas 2-methoxyestradiol caused a concentration-dependent (1 to 100 nmol/L) inhibition of FCS-induced [3H]thymidine and [3H]proline incorporation in COMT-KO SMCs (Figure 1, E and F).
FCS increased cell numbers by 8-fold. After 6 days of treatment, estradiol and 2-hydroxyestradiol inhibited FCS-induced increases in cell number in WT but not COMT-KO SMCs (Figure 2A). 2-Methoxyestradiol blocked FCS-induced increases in cell numbers to a similar extent in WT versus COMT-KO SMCs.
ICI182,780, an ER antagonist, did not attenuate the effects of estradiol on FCS-induced [3H]thymidine incorporation in WT SMCs (Figure 2B). Phenobarbital (CYP450 inducer) enhanced the inhibitory effect of estradiol on cell growth in WT SMCs (Figure 2C). The inhibitory effects of estradiol in WT cells were blocked by 1-aminobenzotriazole (CYP450 inhibitor). In WT SMCs, the inhibitory effects of both estradiol and 2-hydroxyestradiol were blocked by OR-486 (a COMT inhibitor; Figure 2, C and E). 2-Methoxyestradiol inhibited growth to the same extent in both WT and COMT-KO cells, and these effects were not modulated by OR-486 (Figure 2E). The effects of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol on COMT-KO SMCs were not influenced by phenobarbital, 1-aminobenzotriazole, or OR-486 (Figure 2, D and E).
Estradiol protects against injury-induced neointima formation by inhibiting SMC growth.4 Whether the inhibitory effects of estradiol on SMC growth are ER-mediated remains unclear. Estradiol inhibits proliferation of SMCs in injury-induced vascular lesions in mice lacking ERα,1 ERβ,2 and both ERα and ERβ,3 suggesting that the inhibitory effects are ER-independent. An ER-independent mechanism is also supported by the observations that estradiol inhibits injury-induced neointima formation in gonadectomized but not intact male rats, although vascular cells from both express ERs.10 Pharmacological evidence for a role of ERs in mediating the antimitogenic effects of estradiol in SMCs is controversial. ICI182,780, an ER antagonist, was effective in blocking the injury-induced neointima formation in one study11 but not in another.12 ICI182,780 not only binds to ERs but also blocks the metabolism of estradiol to 2-hydroxyestradiol by competing for CYP450s.13 We have demonstrated that ICI182,780 blocks the vascular antimitogenic effects of estradiol only at concentrations that inhibit the metabolism of estradiol to hydroxyestradiols.5,13 The above findings suggest that the antiproliferative actions of estradiol are ER-independent.
Because estradiol is sequentially metabolized to hydroxyestradiols by CYP450s and hydroxyestradiols are methylated to methoxyestradiols by COMT, we hypothesize that methoxyestradiols mediate the antiproliferative actions of estradiol on SMCs via ER-independent mechanisms. Previously, using pharmacological agents to block or induce the conversion of estradiol to hydroxyestradiols and methoxyestradiols, we provided indirect evidence that methoxyestradiols mediate the vascular antimitogenic effects of estradiol.5,13 We found similar evidence for this hypothesis in cardiac fibroblasts14 and glomerular mesangial cells.15
Although use of pharmacological agents provides evidence supporting our hypothesis, direct evidence for the role of methoxyestradiols as mediators of the ER-independent antimitogenic actions of estradiol is lacking. Because COMT is essential to convert hydroxyestradiols to methoxyestradiols, COMT-KO mice provide the ideal model system to directly test our hypothesis.
The fact that estradiol does not inhibit FCS-induced growth of COMT-KO SMCs provides direct evidence that methoxyestradiols mediate the antimitogenic effects of estradiol. The fact that both WT and COMT-KO SMCs expressed ERα/β suggests that the lack of antimitogenic effects of estradiol in COMT-KO SMCs is because of lack of methoxyestradiol formation and not because of absence of ERs. This conclusion is further supported by our finding that WT but not COMT-KO SMCs convert 2-hydroxyestradiol to 2-methoxyestradiol. Also, ICI182,780 fails to block the antimitogenic effects of estradiol in SMCs from WT mice. Our observation that 2-hydroxyestradiol (a 2-methoxyestradiol precursor) inhibits the growth of WT but not COMT-KO SMCs provides further evidence that methoxyestradiols mediate the antimitogenic effects of estradiol and hydroxyestradiols. Our observation that 2-methoxyestradiol is equipotent in inhibiting growth of WT and COMT-KO SMCs is also consistent with our hypothesis. Together, our findings provide strong evidence that methoxyestradiols mediate the antimitogenic effects of estradiol in SMCs via ER-independent mechanisms.
Estradiol is sequentially converted to hydroxyestradiols and methoxyestradiols by CYP450 and COMT, respectively.13 Because both CYP450s and COMT are expressed in SMCs,13 estradiol can be locally converted to methoxyestradiols. In WT SMCs, the inhibitory effects of estradiol are significantly enhanced by phenobarbital, a CYP450 inducer,13 and blocked by 1-aminobenzotriazole, a CYP450 inhibitor.5,13 Moreover, the inhibitory effects of estradiol and 2-hydroxyestradiol are abolished by OR-486 (a COMT inhibitor).13 These findings provide additional evidence that methoxyestradiols mediate the antimitogenic effects of estradiol in SMCs. Because this mechanism mediates the antimitogenic effects of estradiol in SMCs from both humans8 and rats,5 as well as in rat cardiac fibroblasts14 and rat and human glomerular mesangial cells,15 we conclude that this mechanism is well integrated in regulating the biological effects of estradiol in various species/organs.
Unpublished data from our laboratory indicate that testosterone and medroxyprogesterone inhibit conversion of estradiol to 2-hydroxyestradiol. The lack of inhibitory effects of estradiol on injury-induced neointima formation in nongonadectomized rats11 and rats treated with medroxyprogesterone11 may be because of the inhibition of estradiol metabolism.
Our hypothesis predicts that the antimitogenic effects of estradiol may not be mimicked by a wide range of estrogens that are not metabolized to methoxyestradiols. This raises an important issue when comparing the effects of various estrogenic preparations used clinically, such as conjugated equine estrogens.16
The mechanism by which 2-methoxyestradiol inhibits SMC growth has been partially elucidated. 2-Methoxyestradiol binds at the colchicine-binding site of tubulin and disrupts tubulin polymerization, a process essential for cell division. Also, 2-methoxyestradiol blocks SMC growth in both G0/G1 and G2/M phases of the cell cycle, by inhibiting cyclin-D1 and cyclin-B1 expression.17 Moreover, 2-methoxyestradiol inhibits phosphorylation of retinoblastoma protein, ERK1/2 (MAPK),7 and Akt (mitogenic pathways responsible for cell growth) and upregulates Cdk inhibitor p27 (responsible for arresting cells in G1 phase).17
It is feasible that endogenous estradiol could provide sufficient concentrations of 2-methoxyestradiol to inhibit SMC growth in vivo. Circulating levels of estradiol in premenopausal women range between 0.3 and 1 nmol/L, whereas the levels of methoxyestradiols in pregnant women are 30 nmol/L.4 The fact that 1 nmol/L of estradiol is sufficient to inhibit SMC growth via 2-methoxyestradiol suggests that endogenous 2-methoxyestradiol may regulate SMC growth. Moreover, because estradiol is locally synthesized within many tissues, including the blood vessel wall, its levels in the vicinity of SMCs may be higher than circulating levels. Also, whereas our studies used short-term incubations with estradiol, in humans, blood vessels would be exposed to estradiol for years. Thus, the cumulative effects of estradiol via 2-methoxyestradiol may be underestimated by in vitro studies.
In conclusion, using COMT-KO mice, we provide direct evidence that the antiproliferative effects of estradiol are mediated via an ER-independent mechanism that involves the sequential conversion of estradiol to methoxyestradiols. Thus, endogenous methoxyestradiols may protect against cardiovascular disease by preventing vascular SMC growth. Also, because 2-methoxyestradiol is a potent antiangiogenic agent, it may negatively influence vascular plaque formation by inhibiting the growth of capillaries that feed plaques.
This study was supported by the Swiss National Science Foundation, grant 32-64040.00.
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