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The Stehlin Foundation for Cancer Research, 1918 Chenevert St, Houston, TX 77003, USA
(Requests for offprints should be addressed to C S A Markides; Email: cmarkides{at}stehlin.org)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß). The physiological role of this binding remains to be elucidated. |
Introduction |
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The major pathway for oxidative metabolism of E2 in the liver is 2-hydroxylation. Formation of 4-OHE2 occurs as a quantitatively minor pathway, typically less than 15% of 2-hydroxylation (Zhu & Conney 1998). The enzymes responsible for catalyzing this reaction belong to the cytochrome P450 family and include CYP1A2 and the CYP3A families (Kerlan et al. 1992, Lee et al. 2003). 4-OHE2 is formed through a lack of specificity of these enzymes. The most likely function of hepatic estrogen metabolism is to prepare the steroids for excretion.
In contrast to this metabolic profile seen in the liver, 4-hydroxylation is a dominant pathway of catechol estrogen formation in several extrahepatic tissues in both animal models and humans. Selective 4-hydroxylation of E2, with little or no 2-hydroxylation activity, has been observed in human uterine myoma, where 4-OHE2 is the predominant catechol estrogen, formed at rates 5-fold higher than those in the surrounding myometrium (Liehr et al. 1995). CYP1B1 has been identified as a major enzyme catalyzing the 4-hydroxylation of E2. In fact, E2 may be the physiological substrate for human CYP1B1, principally because of its low Km value (0.71 µM) for E2 (Hayes et al. 1996). This specific 4-hydroxylase has been identified in many of those organs of rodents in which chronic estrogen exposure induces malignant or benign tumors: hamster kidney (Kirkman 1959), mouse uterus (Newbold et al. 1990, Newbold & Liehr 1999) or rat pituitary gland (Bui & Weisz 1988). As mentioned above, specific 4-hydroxylation of E2 also occurs in normal or neoplastic human tissues, such as myometrium (Liehr et al. 1995) and breast (Liehr & Ricci 1996).
Selective expression of estrogen 4-hydroxylase activity in target tissues does not inactivate the parent estrogen but may be a mechanism for maintaining hormonal activity in these tissues (Zhu & Conney 1998). Tissue-specific metabolism of E2 is likely a form of differential regulation of estrogenic action and may point to a distinct physiological role for 4-OHE2. For example, 4-OHE2 has been shown to upregulate the uterine expression of lactoferrin in estrogen-receptor (ER)-knockout (ERKO) mice 60-fold over vehicle control, while E2 produced only a doubling of lactoferrin mRNA (Das et al. 1997). This upregulation of lactoferrin by 4-OHE2 was not inhibited by ICI 182,780, an estrogen-receptor antagonist, indicating a pathway independent of both ER and ERß. Also in ERKO mice, 4-OHE2 has been implicated in mammary growth and development (Weisz et al. 1993). Paria et al.(1990) showed that 4-OHE2 plays a definitive role during blastocyst implantation. More-recent data suggest that both and E2 and 4-OHE2 are essential for implantation. E2 prepares the progesterone-primed uterus to the receptive state via interaction with the classical estrogen receptor, while 4-OHE2 makes the blastocyst ‘implantation-competent’ via the generation of prostaglandins (Paria et al. 1998). The 4-hydroxylase activity in the uterus of the pregnant mouse changes drastically as the pregnancy progresses. E2 and progesterone elevate the levels of the specific 4-hydroxylase activity on day 4 of the pregnancy, precisely the time at which implantation occurs (Paria et al. 1990). Also, a surge in the 4-hydroxylase activity was noted in the pig blastocyst on days 12 and 13 of pregnancy, a time that corresponds to blastocyst implantation (Mondschein et al. 1985).
2-Hydroxyestradiol (2-OHE2) and 4-OHE2 possess different physiologic potencies and functions. For example, the uterotrophic potency of 4-OHE2 is close to that of E2, while that of 2-OHE2 is considerably weaker (Barnea et al. 1983, Paria et al. 1990). This is true even though both catechol estrogens have similar binding affinities to ER and ERß, albeit 5–10-fold weaker than that of E2 (Schütze et al. 1994). 4-Methylestradiol, which is incapable of being metabolized to 4-OHE2, is an estrogen agonist with about 25% the hormonal activity of E2 based on relative binding to the ER and induction of the progesterone receptor in MCF-7 cells (Vollmer et al. 1991). Despite this, 4-methylestradiol is incapable of inducing uterine weight gain (Qian & Abul-Hajj 1990, Ball et al. 1983). This indicates that 4-hydroxylation of E2 is necessary for the expression of at least some of the estrogenic effects of E2.
4-OHE2 induces the expression of vascular endothelial growth factor-A (VEGF-A) through a pphosphoinositide 3-kinase-mediated pathway (Gao et al. 2004). Also through a phosphoinositide 3-kinase pathway, independent of described estrogen receptors, 4-OHE2 has been shown to activate the antioxidant-responsive element, which plays a role in gene expression of phase II metabolism enzymes (Lee et al. 2003). It has also been shown to be capable of stimulating the proliferation of human female osteoblastic cells, independently of the two known estrogen receptors (Seeger et al. 2003).
All the above-mentioned data indicate a role for other than that of an estrogenic metabolite 4-OHE2 binding to estrogen receptor(s) (ER and/or ERß), formed solely for facilitating excretion. In an attempt to understand this potential physiological role for 4-OHE2, we have investigated the selective binding of 4-OHE2 to binding proteins other than the described estrogen receptors in wild-type mice.
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Materials and Methods |
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[6,7-3H] E2 (specific activity, 50 Ci/mmol) was obtained from American Radiolabeled Chemicals (St Louis, MO, USA). E2, 4-OHE2 and 2-OHE2 were obtained from Steraloids (Newport, RI, USA). Potassium nitrosodisulfonate was obtained from Aldrich Chemicals (Milwaukee, WI, USA). Hydroxyapatite (HAP) was obtained from Bio-Rad (Hercules, CA, USA). Tris base, EDTA, dithiothreitol, Na2MoO4, KCl and glycerol were all obtained from Sigma Chemicals (St Louis, MO, USA).
Animals
All mice used were non-inbred Swiss NIH high-fertility strain. The source of the animals was the nude mouse colony at the Stehlin Foundation for Cancer Research. Animals used for the study were female mice of various ages, ranging from newborn to 10 months old. They were selected from non-nude animals. In addition, in order to select for animals of precise ages, animals were bred specifically for this study. For this purpose, 10 homozygous dominant females were bred, at any one time, with five homozygous dominant males. Their offspring were killed at 1, 2, 3, 4, 8 or 16 weeks of age. Experimental groups consisted of tissue collected and pooled from several animals. Six tissue pools were used to accumulate the data from uterus and three from lung, while all other data were collected in duplicate. Animals did not undergo any treatments and were not administered any drugs at any point before being killed. All animal handling and euthanasia was conducted in accordance with Institutional Animal Care and Use Committee-approved procedures.
Synthesis of radiolabeled 4-OHE2
[6,7-3H]4-OHE2 was synthesized from [6,7-3H]E2 according to a modified procedure detailed by Gelbke et al.(1973). The resulting radiolabeled catechol estrogens were purified by reversed-phase HPLC using a C18 column. The solvent gradient of aqueous buffer (75 mM citric acid/25 mM ammonium acetate)/acetonitrile/methanol from 80:15:5 to 30:50:20 (by vol.) at 1 ml/min over 50 min resolved the products as follows: 4-OHE2 at 31.2 min, 2-OHE2 at 32.3 min and E2 at 38.4 min. Solutions of the pure 3H-labeled 4-OHE2 were stored in the presence of 10% acetic acid at –80 °C until used.
Preparation of the extract
Animals were killed and their uteri removed. The uteri were washed in ice-cold 1 x PBS (4% NaCl, 1% KCl, 0.47% Na2HPO4, 0.1% KH2PO4, pH 7.3) and subsequently homogenized in about 3 vol. of ice-cold KTEDMG buffer (300 mM KCl, 1 mM sodium EDTA, 10 mM Tris base, 1 mM dithothreitol and 10 mM Na2MoO4, pH 7.3; made up in a 10% glycerol solution) containing protease inhibitors. Gentle homogenization was carried out in glass homogenizers to prevent denaturation of the proteins. Cytosolic fractions of the uterine homogenate were prepared by ultracentrifugation (40 000 r.p.m. (165 000 g) at 4 °C for 1 h) in a Beckman L7–65 ultracentifuge. Total protein concentrations of the fractions were determined using the Bradford protein assay.
Binding assay
For total binding, the total cytosolic fraction resulting from each experimental group was normalized to 1 mg total protein/ml, divided into six aliquots (200 µl) and incubated with 2.0 nM radiolabeled [6,7-3H]4-OHE2. For the determination of non-specific binding, another set of tubes received a 400-fold molar excess (800 nM) of unlabeled or 4-OHE2, E2 or 4-OHE2 plus E2 in addition to the radiolabeled steroid. After incubation at 4 °C for 18–20 h (overnight), bound and unbound radioligand were separated by using HAP: 400 µl of a 1:1 suspension of HAP in TED buffer (10 mM Tris base, 10 mM sodium EDTA and 1 mM dithiothreitol, pH 7.3) were added to the incubation volume (200 µl) and the mixture incubated on ice for about 15 min, vortexing every 5 min. The HAP was then spun down to a tight pellet (3000 r.p.m. for 10 min at 4 °C). The pellet was washed three times with 2 ml aliquots of ice-cold TED containing 1% Tween 80. Bound steroid was extracted by resuspending the HAP in 2 ml ethanol (100%) for 15 min at room temperature, vortexing occasionally. The HAP was spun down and an aliquot (1 ml) of the supernatant was counted in a liquid scintillation counter.
Unlabeled saturation assays
These experiments were conducted in a similar fashion to the binding assay except that instead of the 400-fold molar excess of or E2 or 4-OHE2, increasing concentrations of unlabeled 4-OHE2 or 2-OHE2 were used to generate a saturation curve with unlabeled reactants.
Hormonal specificity studies
Hormonal specificity studies were conducted in a similar manner as above, using a 400-fold molar excess of the selected steroid in the presence or absence of a 400-fold molar excess of E2, to rule out cross-binding to estrogen-binding sites. Since ethanol is used to denature the proteins and extract the radioligand, binding observed is reversible binding and not the result of a covalent bonding of the catechol to the protein, as described previously (Abul-Hajj & Cisek 1988). Specific binding is expressed as (total binding – non-specific binding) and is expressed as fmol of [6,7-3H]4-OHE2 bound per mg cytosolic protein.
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Results |
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). In the presence of excess unlabeled 4-OHE2 the binding recorded was 82.1 ± 1.7 fmol/mg protein, but in the presence of excess unlabeled E2 the binding decreased only to 214.6 ± 9.4 fmol/mg protein. The difference between displacements of 3H-labeled 4-OHE2 by unlabeled E2 and unlabeled 4-OHE2 was taken as evidence of specific binding to a 4-OHE2-binding protein. This binding to uterine protein of 3-week-old animals served as a positive control in more than a dozen independent experiments and consistently yielded the specific 4-OHE2 binding shown in Fig. 1. There was little or no difference between binding in the presence of excess unlabeled 4-OHE2 and 4-OHE2 plus E2, indicating binding of the catechol estrogen to a specific binding protein in addition to any ER and/or ERß binding. 4-OHE2 binds to the ER with a dissociation constant (Kd) of 0.21 nM (Barnea et al. 1983, Schütze et al. 1994) and so a portion of the total binding of the radioligand would have to account for such binding. The data derived from 3-week-old animals were used as a gold standard, as the results were verified in more than a dozen independent experiments.
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), this binding was entirely displaced by E2. Binding likely includes the relatively low levels of ER identified previously (Shigeta et al. 1996) by Northern analysis in this tissue and possibly other binding proteins.
When increasing concentrations of unlabeled 4-OHE2 (0.1–500 nM) were incubated with a fixed concentration of 4-OHE2 (2 nM), a sigmoidal binding-inhibition curve was observed as the radioligand was gradually displaced from its specific binding sites by the increasing concentrations of the unlabeled ligand, as seen in Fig. 2A. If viewed as a Scatchard plot, two distinct binding components can be observed (Fig. 2B). In order to distinguish the binding of 4-OHE2 to the ER(s) from its specific binding to its own putative receptor, the unlabeled saturation study was also conducted in the presence of 800 nM E2. In the presence of this saturating concentration of E2 the displacement curve of 4-OHE2 was much steeper, as can be seen in Fig. 2A. If viewed as a Scatchard plot, only a single binding component can be seen (Fig. 2B). The Kd value for the specific binding of 4-OHE2 was calculated from these and other similar experiments to be 11.8 ± 2.1 nM. The Kd calculated from the high-affinity component of the Scatchard plot was 0.29 ± 0.03 nM. This corresponds to previously reported Kd values of 4-OHE2 for the ER in MCF-7 cells (Schütze et al. 1994).
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, specific 4-OHE2 binding – the difference in displacement of the radioligand by E2 and 4-OHE2 – as described above, is age-dependent. Levels were low, barely detectable, during the first weeks of life, and then reach a peak of 159.9 ± 12.2 fmol/mg protein at 4 weeks (puberty occurs in the mouse around 3–4 weeks of age), before returning to relatively low levels at later ages.
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). Liver, kidney and whole brain of 4-week-old mice were also examined, but showed no appreciable specific binding of 4-OHE2.
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. As described above, 800 nM E2 partially displaced radiolabeled 4-OHE2 from its binding sites, while unlabeled 4-OHE2 was much more efficient in this respect (Fig. 5A). Of the compounds tested, only 2-OHE2, 4-hydroxytamoxifen, 6-hydroxyestradiol and 11ß-hydroxyestradiol showed any signs of being able to displace radiolabeled 4-OHE2 from its binding sites. The affinity of 2-OHE2 for the estrogen receptor has been established previously (Barnea et al. 1983, Schütze et al. 1994). 4-Hydroxytamoxifen has been shown previously to have significant affinity for the estrogen receptor (Foster et al. 1985) and 6- and 11 ß-hydroxyestradiol have been shown to exert estrogenic effects or to be significant estrogenic metabolites (Dehennin et al. 1984, Segaloff & Gabbard 1984). Our data are consistent with binding of these compounds to E2-binding sites, but not to 4-OHE2-binding sites.
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). This indicates that the displacements caused by the steroids tested were from estrogen-specific binding sites, not 4-OHE2-specific sites. The only exception to this was 2-OHE2. At the concentration tested (800 nM), 2-OHE2 could displace 4-OHE2 from its specific binding site as well as from its binding to E2-binding sites. To further examine the binding of 2-OHE2 to the specific 4-OHE2-binding site, a parallel study was conducted using the same amounts of radioligand and increasing concentrations of either unlabeled 4-OHE2 or unlableled 2-OHE2. The results can be seen in Fig. 6, which shows 2-OHE2 to be a competitive inhibitor of 4-OHE2 binding, albeit with an inhibition constant (Ki) 10-fold greater than the Kd value of 4-OHE2 (98.2 ± 12.6 nM).
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Discussion |
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is known (Barnea et al. 1983), our data demonstrate protein binding distinct and different from binding to ER. Total binding of 4-OHE2 likely includes binding to ER, ERß, a putative 4-OHE2 receptor and possibly other binding proteins. The difference in displacement of 3H-labeled 4-OHE2 by unlabeled 4-OHE2 and unlabeled E2, however, is clear evidence of the existence of a specific binding protein, which may stimulate a hormonal signaling pathway distinct and different from that of E2. Binding of 4-OHE2 to the ER(s) has been measured in the past (Schütze et al. 1994) during investigation of the physiological role of catechol metabolites of E2. That study corroborated previous results (Barnea et al. 1983) that 4-OHE2 binds to the ER, but dissociates more slowly than E2. Therefore hydroxylation of E2 may be a mechanism for prolonging estrogenic action in certain tissues (Zhu & Conney 1998). Schütze et al.(1994), however, observed higher ‘non-specific binding’ when using radiolabeled 4-OHE2 rather than E2. Since they were using diethylstilbestrol to displace the radioligand, this higher value for non-specific binding could be attributed to binding of the radioligand to a 4-OHE2-specific binding site, such as the one described in this study, from which it could not be displaced by diethylstilbestrol.
In fact, as described in this text, none of the steroids tested except 2-OHE2 were capable of displacing radiolabeled 4-OHE2 from its specific binding site, even when using a 400-fold molar excess. The 10-fold lower affinity of 2-OHE2 for the 4-OHE2-specific binding site indicates that the binding of 2-OHE2 is not a hormonal effect. Also, even though the Km values of both catechol estrogens for catechol-O-methyl transferase are similar, 2-OHE2 is much more rapidly methylated than 4-OHE2 (Roy et al. 1990). This results in 2-OHE2 levels that are universally low (Emons et al. 1987), nowhere close to 98 nM, which we have determined as its Kd for the 4-OHE2-specific binding site.
Philips et al.(2004) recently described specific 4-OHE2 binding in ERKO mice. The Kd they report however, are a full order of magnitude lower than the one reported in this paper in 3-week-old wild-type mice. This difference could indicate a different binding dynamic in the presence of ER or even the existence of a totally different binding protein. The decline in the level of specific 4-OHE2 binding in mice aged more than 4 weeks old (Fig. 3) could also point to a different protein being expressed during puberty.
Catechol estrogens are easily oxidizable to the semiquinone and quinone forms, which may bind covalently to proteins. The protein binding described by us, however, is different from covalent binding, as evident from the reversal of binding by the ethanol wash. Moreover, the lack of binding in mouse uterus at 1 week of age or in other organ sites indicates specific reversible binding and not non-specific, covalent binding, which would be expected to occur in any tissue.
The existence of the specific binding indicates that 4-OHE2 has physiological activity, as indicated previously by its role in blastocyst implantation (Paria et al. 1990, 1998) and lactoferrin gene expression (Das et al. 1997). Both these events take place in the uterus, an organ where we detect binding to a specific protein. The specific binding of 4-OHE2 described in this text could account for this behavior of 4-OHE2. Circulating levels of 4-OHE2 are undetectable. Within hormonally active tissues, however, such as human breast cancer, high levels in the nanomolar range have been detected (Yue et al. 2003). This is due to the fact that in the blood serum enzymes, such as catechol-O-methyl transferase, rapidly metabolize 4-OHE2, while these enzymes are not necessarily present in tissues where 4-OHE2 is formed in situ. The distribution of CYP1B1, the isozyme responsible for selective 4-hydroxylation of E2, is known (Shimada et al. 1996) and could be a good indicator of tissues that express 4-OHE2-specific binding.
Our data indicate that 4-OHE2 may be formed in the uterus by CYP1B1 (Shimada et al. 1996) as a signaling molecule. The existence of this estrogen 4-hydroxylase in organs other than uterus, such as lung (Shimada et al. 1996), point towards an as-yet unknown role of this steroid in other organ sites. The exact nature of the binding protein as well as the more-specific role of 4-OHE2 in the lung and other organs remain to be elucidated.
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Acknowledgements |
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Funding |
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This work was funded in part by charitable donations to the Stehlin Foundation for Cancer Research and in part by NIH grant NCI 74971. The authors declare that there are no conflicts of interest in this work.
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References |
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Received in final form 3 December 2004
Accepted 27 January 2005
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, right-hand y axis) and in the absence of E2 (•, left-hand y axis). (B) The same data presented as a Scatchard plot.
