Molecular and Cellular Determinants of Estrogen Receptor α Expression

Critical roles for estrogens in growth and development and in pathological conditions of bone, breast, and uterus are well established. Estrogens and estrogen receptor modulators bind to estrogen receptor α (ERα) and/or ERβ to form discrete molecular complexes that exert pleiotropic tissue-specific effects by modulating the expression of target genes. Ligand-bound ER functions as a key transcription factor in various molecular pathways, and modulation of ER expression levels is important in determining cellular growth potential. Recent advances have begun to illuminate the mechanisms by which cells control ER expression. Kos et al. reviewed the genomic organization of the human ERα gene promoter region (31). The human ERα gene, located on chromosome 6, was cloned in 1986 and spans approximately 300 kb; its eight coding regions are transcribed from at least seven promoters (31). Considering the complexity of the ER promoter, it is not surprising that numerous factors affect ERα expression. The aim of this paper is to review the molecular mechanisms by which various cellular factors modulate ERα expression. We have focused on highlighting the major players, including effectors of chromatin structure, hormones, and other relevant agents, and limited our discussion to findings in human systems. There are vast qualitative and quantitative data regarding whether or not, and to what extent, ERα is expressed in normal and neoplastic tissues; this topic is beyond the scope of this review and will not be covered. Moreover, little is currently known about the role of specific transcription factors in ER expression, and these factors will be mentioned only briefly. Henceforth, the terms ERα and ER will be used interchangeably; expression of ERβ will not be discussed.

EFFECTORS OF CHROMATIN STRUCTURE

Epigenetic regulation is an important mechanism by which cells modulate the expression of a variety of genes. Down-regulation of ERα gene expression has been attributed to methylation in a variety of tissues including colon, blood, lung, heart, prostate, and ovary (27-29, 35, 42, 44, 45, 52). This mechanism has been most extensively studied in breast cancer cell lines and tissue to explain the loss of ER expression and acquired hormone resistance associated with this disease. Studies have consistently demonstrated that, whereas normal breast tissues and ER⁺ breast cancer cell lines lack methylation of the ER gene, ER⁻ breast cancer cell lines and tumors display extensive methylation (35, 44). DNA methyltransferase 1 (DNMT1) levels and activity are increased 2- to 10-fold in ER⁻ breast cell lines, in agreement with the levels of methylation observed in these cells (44). The methylation inhibitor 5-aza-2′-cytidine (5-aza-dC) sequesters DNMT1 to 5-aza-dC-substituted DNA and inhibits DNMT1 action (17, 30). The use of 5-aza-dC resulted in demethylation of the ER CpG island and reexpression of ER mRNA and protein (18). Thus, methylation was established as playing a direct role in the mechanism of ER transcriptional regulation. Lapidus et al. utilized a methylation-specific PCR assay to map the ER CpG island (35). The degree of methylation of the ER gene in breast tumors exhibited an inverse association with ER status, with the lowest levels of methylation seen in ER⁺/progesterone receptor-positive (PR⁺) tumors, intermediate levels seen in ER⁺/PR⁻ tumors, and the highest levels seen in ER⁻/PR⁻ tumors. Interestingly, 35% of the ER⁺/PR⁺ tumors showed substantial methylation. Due to the heterogeneous nature of ER expression in breast cancers, it has been speculated that ER methylation in ER⁺ tumors might serve as a predictor of which tumors may recur as ER⁻ tumors or those that will develop resistance to hormonal therapy (35).

In contrast to breast carcinomas, the role of methylation in ER down-regulation in endometrial tumors is less well defined. Two separate groups failed to determine a significant correlation between a lack of ER expression and hypermethylation of the ER gene (24, 41). However, two additional studies concluded that methylation was associated with ER status in endometrial carcinomas (51, 55). Importantly, Sasaki et al. analyzed the methylation status of three isoforms generated from promoters A, B, and C (51) (as defined in reference 22). Expression of isoforms ER-A and ER-B was observed in endometrial cancer cell lines, unlike isoform ER-C, which was not expressed in any cell lines tested. Whereas ER-A and ER-B were not methylated in cancerous or normal tissues, ER-C was methylated in 94% of tumor tissues. Thus, the ER-C transcript was shown to be inactivated in endometrial carcinomas, where its lack of expression was associated with methylation.

The incidence of ERα gene methylation has also been linked to the development of heart disease. In the cardiovascular system, estrogen operates through the ER to increase the synthesis of nitric oxide, which promotes blood vessel dilation and prevents inflammation caused by platelets (19). Post et al. discovered an increased frequency of ER methylation in coronary atherosclerotic plaques when compared to normal tissue (45). Notably, ER methylation increased with age in the atrium and was observed principally in smooth muscle cells.

Numerous studies have postulated that methylation of the ER gene is not the sole mechanism for the inactivation of its expression (Fig. 1). In fact, there is increasing evidence that gene silencing by methylation also involves the assistance of histone deacetylases (HDAC) which remove acetyl groups from lysine residues on histones H3 and H4, resulting in the formation of a compact structure that diminishes gene activity (reviewed in reference 40). Yang et al. treated a panel of ER⁻ breast cancer cell lines with trichostatin A (TSA), an HDAC inhibitor; ER mRNA was reexpressed in these cells in a time- and dose-dependent manner, with up to a fivefold increase in ER expression observed (69). However, the ER CpG island remained methylated with ER mRNA levels induced to only 1 to 10% of those seen in MCF7 and T47D ER⁺ cell lines (69). Additional studies have demonstrated that treatment of ER⁻ cells with TSA and 5-aza-dC results in a synergistic effect on ER reexpression (5). One group observed a 300- to 400-fold induction of ER mRNA associated with increased acetylation of histones H3 and H4, decreased levels of DNMT1, and partial demethylation of the ER CpG island (70). If ER⁻ cells were treated with either 5-aza-dC or TSA alone, however, 5-aza-dC only weakly activated ER expression, whereas TSA did not activate ER. Chen et al. established a clonal cell line derived from T47D cells (T47D:C4:2) that exhibited a loss of ER expression and responsiveness to hormones due to its maintenance in estrogen-free medium (7). Importantly, the ER CpG island in C4:2 cells was not methylated, indicating that methylation was not responsible for ER silencing in these cells. It was thus hypothesized that other steps may precede methylation, including a loss of transcriptional activators or the presence of factors that block the activity of methyltransferases (7).

FIG. 1.

Transcriptional regulation of the ER gene by epigenetic mechanisms. The ER gene is transcriptionally regulated by epigenetic mechanisms including methylation and acetylation. The methyltransferase DNMT1 transfers methyl groups (circles) to cytosine bases located within CpG islands in exon 1 and upstream regulatory regions of ER, thereby inhibiting transcription. Transcriptionally active ER is characterized by the presence of acetyl groups (wavy lines) on histones H3 and H4, which promotes an open, accessible chromatin conformation. Upon removal of the acetyl groups by HDAC, the chromatin becomes condensed where ER is now in a transcriptionally inactive state. The DNMT inhibitor 5-aza-dC and the HDAC inhibitor TSA work synergistically to allow the reexpression of ER in cells with methylated DNA and acetylated histones.

Two transcription factors, ER factor 1 (ERF-1) and ER-B factor 1 (ERBF-1), which regulate ER expression, potentially in conjunction with or as an alternative to methylation in breast cancer cell lines and tissues, have been described previously (13, 54, 71). ERF-1 has been shown to transactivate promoter A, whereas ERBF-1 is necessary for transcription from promoter B (defined in reference 71). Overexpression of ERF-1 in human mammary epithelial cells resulted in the generation of a DNase I-hypersensitive site in the untranslated region of exon 1 (54). Such sites are associated with regions of open chromatin conformations, thereby indicating that ERF-1 can not only transcriptionally regulate the ER gene but can also alter its chromatin structure (54). Yoshida et al. showed a negative correlation between the methylation status of both promoters A and B and ER expression (71). Upon demethylating treatment, no transcription from either promoter B or promoter A was seen in ER⁻ MDA-MB-231 cells, which do not express ERBF-1 or ERF-1. However, promoter A transcripts, but not promoter B transcripts, were induced in ER⁻ BT-20 cells. ERF-1 is expressed in this cell line, in contrast to the lack of expression of ERBF-1. Thus, it has been suggested that a loss of these factors may be important in preventing the reexpression of ER (71).

Another potential modulator of ER expression is mitogen-activated protein kinase (MAPK) kinase (MAPKK MEK), a downstream effector of growth factor signaling. Oh et al. recently demonstrated that hyperactivation of MAPKK in MCF7 cells leads to reduced levels of ER, whereby the inhibition of MAPKK activity led to its reversible up-regulation (43). Additionally, treatment of ER⁻ breast cancer cell lines with 5-aza-cytidine resulted in both increased ER expression and a 9.4-fold decrease in MAPKK activity. If the MEK inhibitor U0126 was also added to the treated cells, MAPKK activity was further reduced 4.4-fold and ER levels increased by an additional 1.5-fold. Thus, these data provide evidence that the inactivation of MAPKK, together with the demethylation of the ER gene, promotes the reexpression of ER.

HORMONAL AND GROWTH FACTOR PATHWAYS

It is important to consider hormonal and growth factor effects on ER expression since one mechanism by which hormones and growth factors regulate the growth of various tissues is by modulating levels of transcription factors, such as the estrogen receptor, that then affect transcription of other genes. Signals transduced by the estrogen receptor affect the growth and metabolism of estrogen-responsive tissues; therefore, the level of ER transcription is an important determinant of cellular growth potential. Much of the research that has elucidated the mechanism of hormonal control of ER expression focuses on understanding how each hormonal system relates to the pathogenesis and pathophysiology of neoplasia, particularly of estrogen-dependent tumors. Table 1 summarizes the effects of the hormones and hormone receptor modulators, whose molecular mechanisms of action are discussed below, on ERα expression, while Fig. 2 graphically depicts the hormonal pathways involved.

TABLE 1.

Effects of hormones, hormone receptor modulators, and growth factors on ER expression

Agent	Class/pathway	Tissue/cell linea	ER mRNA	ER protein	Reference(s)
Estradiol	Estrogen	MCF-7	Increase	Decrease	37, 49
		Endothelium	Increase	Decrease	26, 64
		Hypothalamus	Increase	Increase	58
		Osteobalst	Increase		15, 16
ICI 182 780	Estrogen antagonist	MCF-7	No effect	Decrease	4
Tamoxifen		MCF-7	No effect		67
		Uterus		Decrease	9
Raloxifene	SERM	MCF-7	Increase		67
ORG 2058	Progesterone agonist	T47D	Decrease		53
R5020	Progesterone agonist	T47D and MCF-7		Decrease	1, 53
RU486	Progesterone antagonist	T47D and uterus		Increase	39, 53
Danazol	Progestogen/estrogen antagonist	PBMC	Decrease	Decrease	20, 63
Flutamide	Androgen antagonist	Prostate		Increase	34
1,25(OH)2D3	Vitamin D	MCF-7	Decrease	Decrease	58, 62
EB-1089	Vitamin D agonist	MCF-7	Decrease	Decrease	62
KH-1069	Vitamin D agonist	MCF-7	Decrease	Decrease	62
Ro 27-0574	Vitamin D agonist	MCF-7	Decrease	Decrease	62
Ro 23-7553	Vitamin D agonist	MCF-7	Decrease	Decrease	62
hCG	Gonadotropin/PKA	Ovary	Decrease	Decrease	8
EGF	Growth factor/PKB	MCF-7	Decrease	Decrease	11, 59
D-Ala-GnRH	GnRH agonist/PKC	Ovary	Decrease	Decrease	8
Insulin	Insulin/IGF-1	MCF-7		Increase	2
IGF-I	Insulin/IGF-I	MCF-7	Decrease	Decrease	60

^aPBMC, peripheral blood mononuclear cells.

FIG. 2.

Hormonal pathways involved in regulation of ER transcription. (A) Nuclear hormone effects on ER transcription. ER_AG, estrogen receptor agonist; ER_ANT, estrogen receptor antagonist; PR_AG, progesterone receptor agonist; T, testosterone; AR, androgen receptor; VDR_AG, vitamin D receptor agonist; RXR, retinoid X receptor. (B) Protein hormone effects on ER transcription. HCG, human chorionic gonadotropin; INS, insulin; ↓ and ⊥, stimulation and inhibition within the nucleus, respectively.

NUCLEAR HORMONAL PATHWAYS

The human breast tumor cell line MCF7 has been used as a model of ERα action. Early reports of estradiol-induced “ER processing” in MCF7 cells suggested that agonist-induced receptor regulation also occurred in human breast ductal epithelium (25). Most recently, estradiol-induced ERα turnover mediated by the ubiquitin ligase 26S proteasome systems has been proposed as an integral part of the estrogen signaling pathway. In this model, rapid estradiol-induced ERα degradation is linked to ERα-mediated transcriptional responses (37, 49). This model predicts that ERα levels in mammary epithelium are maintained by new receptor synthesis.

Human vascular endothelial cells express ERα and contribute to an important vascular response to estradiol, the local generation of nitric oxide. Rapid proteasome-dependent down-regulation of ERα receptors was observed in human vascular endothelial cells in response to estradiol and was followed by the transcriptional activation of the ERα gene 4 h later and the repletion of ERα protein levels by 6 h (26, 64). These results indicate that acute degradation of ERα followed by an estradiol-dependent transcriptional activation of ERα mRNA is a general estradiol response.

Long-term exposure to estradiol also regulates steady-state ERα levels, and the clearest evidence for this type of control stems from investigations of nonreproductive estrogen target tissues including liver, brain, and peripheral nervous tissue. Most studies showed that estradiol maintains these tissues in an estrogen-responsive condition by up-regulating steady-state ERα expression levels. This view is supported by the presence of estrogen response elements in many of the ERα gene promoter regions (16). The ERα protein expressed in liver is transcribed primarily from the estrogen-inducible promoter E located 150 kb 5′ of the ERα translation initiation site. The splicing of DNA sequences from region E onto the ERα generates an ERα mRNA possessing a unique 5′ untranslated region with the potential for liver-specific regulation. Alternative promoter utilization as a means of transcriptional regulation of ERα gene expression promises to be a key factor in the tissue-specific regulation of ERα mRNA and was recently reviewed (48).

Though ERα is widely distributed in the brain, the hypothalamus is a brain region particularly enriched in ERα, and data obtained from this region illustrate the complexity of ERα regulation within the central nervous system. Within the human hypothalamus, the distribution of ERα is concentrated in the band of Broca, the medial mamillary nucleus, the medial preoptic area, the paraventricular nucleus, the lateral hypothalamus, the suprachiasmatic nucleus, and the ventromedial nucleus. Sex differences in ERα levels were observed in these nuclei, implying that estrogens and/or androgens regulate human hypothalamic ERα levels in a nucleus-specific manner (32). Specific evidence for estradiol-mediated up-regulation of ERα levels in the human suprachiasmatic nucleus (controlling circadian functions) has been shown previously (33). These few examples reveal that in the central nervous system, independent ERα gene regulation takes place in different juxtaposed cell types.

ERα is essential to normal bone metabolism and mediates the ability of estradiol to sustain and stimulate bone mineral density in both males and females (57). Studies of primary human osteoblast cell cultures revealed that the regulation of ERα gene expression in bone is tissue specific. As discussed previously, seven distinct ERα promoter regions are dispersed between +163 and −151 kb of the ERα transcription initiation sites (31). In osteoblasts, ERα transcription is initiated at a far 5′ upstream promoter F, ∼117 kb distant from the initiation sites A and C utilized in the human breast cancer cell line MCF7 and primary human aorta smooth muscle cells (14). Although promoter F lacks an estrogen response element consensus sequence common to the other ERα promoter regions, its expression is up-regulated by estradiol-ERα, suggesting that transcriptional regulation of the ERα gene in bone is unique (15, 16). Human osteoblastic cells express high levels of the ERα splice variant ERα46. The ERα46 variant lacks the A/B regions of the N-terminal domain (AF-1) of the full-length ERα66 gene because a splice acceptor in exon 2 rather than exon 1 is utilized (14). The signals controlling the alternative RNA processing of ERα are as yet unclear. However, ERα46 is biologically active and modulates tissue responses to estradiol through the formation of homodimers, heterodimers with ERα66, and heterodimers with ERβ.

The regulation of ERα by pharmacologic agents, phytoestrogens, and xenoestrogens has therapeutic and pathological implications (3). Although changes in ER levels in response to these various ERα ligands are undergoing study and characterization, there is still relatively little information regarding their ability to regulate ERα transcription. Down-regulation of ERα protein levels in response to the pure estrogen antagonist ICI 182,780 (Fulvesant) and other selective estrogen response modifiers (4-hydroxytamoxifen and GW7604, a carboxylated tamoxifen derivative) was studied in human breast tumor cell lines. Increased degradation of ligand-bound ERα occurred via ubiquitination and 26S proteasomal degradation pathways in the rank order ICI 182,780 ≫ estradiol ≫ GW7604 > 4-hydroxytamoxifen. A critical observation that distinguishes the pure estrogen antagonist ICI 182,780 from the potent ERα agonist estradiol is that ICI 182,780 fails to elicit a compensatory stimulation of ERα mRNA (4). Thus, ICI 182,780 caused a sustained depletion of ERα in mammary tumor cells, a therapeutically advantageous condition that precluded further estradiol-stimulated proliferation. Danazol, an antiestrogenic compound in long-term clinical use, has been shown to decrease ER expression in a variety of tissues (20, 63). Most notably, decreased ER mRNA and protein levels were found in the peripheral blood mononuclear cells of patients who were treated with danazol for endometriosis, and a run-on assay revealed that danazol decreased ER transcription (20).

Tamoxifen and raloxifene are clinically important selective estrogen receptor modulators (SERMs); neither drug down-regulated ERα levels in MCF7 cells growing in tissue culture, and in fact, tamoxifen stabilized the nuclear form of ERα. Tamoxifen treatment for 48 h caused no change in ERα mRNA levels in MCF7 cells, but raloxifene stimulated ERα mRNA two- to threefold (67). The basis for the increase in ERα mRNA in response to raloxifene is not known, and other SERMs showed a similar but more modest effect. Although in vivo administration of tamoxifen generally resulted in lower ER levels, there is no evidence that transcriptional regulation contributed to these effects. The in vivo actions of tamoxifen reflect a composite of its mixed agonist-antagonist activity and metabolism to 4-hydroxytamoxifen. When given to women for breast cancer treatment, tamoxifen can elicit a transient tumor “flare” response reflective of its estrogen agonist activity; however, chronic tamoxifen administration to women antagonizes breast tissue growth. In a study of human breast cancer patients previously treated with tamoxifen, endometrial hyperplasia and benign endometrial polyps were sampled immunochemically for ER levels, and ER levels were reduced compared with healthy, age-matched controls (9). The basis of an emerging model to predict the action of SERMs on ER turnover is the idea that different ligand-induced ERα conformations modulate the level of receptor ubiquitination and thus proteasomal degradation rates (46, 65, 68). This concept may drive the development of SERMs that selectively stabilize ERα or target it for destruction.

In contrast to the variable effects of estrogens, most studies consistently demonstrate that progesterone and progesterone agonists down-regulate ER expression. The PR agonist ORG 2058 decreased ER mRNA expression by 40% in breast cancer-derived T47D cells, while R5020, another PR agonist, decreased ER protein levels by 50% in T47D cells and to a lesser extent in MCF7 cells (1, 53). In support of these findings, the PR antagonist mifepristone (RU486) increased ER protein expression by two- to fourfold in T47D cells (53). Moreover, a single 200-mg dose of RU486 administered to nine regularly menstruating women two days after the luteinizing hormone peak resulted in intense nuclear staining for ER and PR in both endometrial gland and stromal cells, while faint or absent staining was noted in control cycles (no RU486) in the same women (39).

Androgens also modulate ER expression; prostatic stromal cells from patients with prostate cancer who were treated with androgen deprivation therapy had increased ER detected immunohistochemically compared to untreated patients, suggesting that androgens suppress ER expression in human prostatic stromal cells (34). These findings raise the intriguing possibility that androgens mitigate the breast cancer-enhancing effects of estrogens.

Finally, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active form of vitamin D, is a potent inhibitor of breast cancer cell growth (10, 38, 56, 66). Swami et al. found that 1,25(OH)₂D₃ reduced MCF7 cell proliferation in a dose-dependent manner; furthermore, this antiproliferative effect correlated significantly with 1,25(OH)₂D₃-mediated ER suppression as measured by an E₂ binding assay (62). In the same study, Northern and Western blot analyses revealed a transient decrease in ER mRNA and protein levels with nadirs at 24 and 48 h, respectively. Moreover, a transcription run-on assay confirmed a transient 1,25(OH)₂D₃-dependent depression of ER gene transcription, while cycloheximide did not prevent the decrease in mRNA, implying that ongoing protein synthesis is not required for 1,25(OH)₂D₃-mediated ER down-regulation. Finally, 1,25(OH)₂D₃ completely prevented the E₂-mediated increase in functional PR and BRCA1 protein expression, suggesting that these are biologically relevant findings (62). Stoica et al. identified a vitamin D response element within the ER promoter; a series of experiments by that group, in which MCF7 cells were treated with 10 nM 1,25(OH)₂D₃, revealed a 60% decrease in ER gene transcription, a 50% decrease in steady-state ER mRNA levels, and a 50% decrease in ER protein levels (58). Stoica et al. also demonstrated a 1,25(OH)₂D₃-induced decrease in the number of ER binding sites, while the binding affinity of estradiol for the ER remained unchanged (58). Importantly, a mutation of the vitamin D response element inhibits the 1,25(OH)₂D₃ effects. Further evidence of a 1,25(OH)₂D₃-mediated decrease in ER expression levels includes correlative studies of EB-1089, KH-1060, Ro 27-0574, and Ro 23-7553, vitamin D analogues that suppress ER levels even more potently than 1,25(OH)₂D₃ (62).

PEPTIDE HORMONES AND GROWTH FACTORS

Human chorionic gonadotropin (hCG) decreases ER expression. Chiang et al. extensively studied hormonal regulation of ERα expression in human granulosa-luteal cells (hGLCs) in vitro (8). First, using a semiquantitative reverse transcription-PCR protocol, they were unable to detect changes in ER expression levels in hGLCs left to spontaneously luteinize in culture for up to 10 days. They then demonstrated a time- and dose-dependent decrease in ER mRNA levels in hGLCs treated with hCG for 24 h; however, the effect was not noted until the cells had been in culture for 7 days but persisted until day 10. Importantly, treatment of hGLCs with 8-bromo-cyclic AMP or forskolin also resulted in decreased ER mRNA expression, suggesting that hCG may activate the protein kinase A (PKA) signaling pathway. In addition, cotreatment of hGLCs with hCG plus a specific PKA or adenylate cyclase inhibitor prevented the decrease, providing further support for this theory. Finally, Western blot analysis confirmed that hCG, 8-bromo-cyclic AMP, and forskolin treatment results in lower ERα protein levels in hGLCs.

Overexpression of erbB2 (HER2) in advanced breast cancer is associated with hormone unresponsiveness, implicating the epidermal growth factor (EGF) pathway in ER regulation. Careful study of MCF7 cells treated with EGF revealed a decrease in ER binding sites without a change in ER binding affinity (11, 59). These results were extended to show that EGF decreased ER protein and mRNA, and this was due to a decrease in ER gene transcription (59). Tyrphostins and wormanin blocked the EGF effects, suggesting that phosphatidylinositol 3-kinase and PKB (Akt), the downstream targets of phosphatidylinositol 3-kinase, may mediate the EGF response (59).

The modulation of ER expression by gonadotropin-releasing hormone (GnRH) may depend on the tissue studied and the mode of delivery. A time- and dose-dependent decrease in ER mRNA levels was noted when hGLC cells were treated continuously for 24 h with the GnRH agonist d-Ala⁶-GnRH (GnRHa) (8). Interestingly, the decrease was noted on day 7 of culture, but no decrease was detected on day 1 or on day 10, indicating specific temporal regulation. Cotreatment of hGLCs with GnRHa plus the specific PKC inhibitor GF109203X inhibited the decrease in ER mRNA, suggesting that GnRH signals via the PKC pathway in the ovary. Western blot analysis in hGLCs revealed that GnRHa treatment also leads to lower ER protein levels.

Insulin and insulin-like growth factor I (IGF-I) signal via a complex cascade of proteins, including insulin substrate 1 (IRS-1), and have pleiotropic effects on ER biology. IGF-I and IRS-1 promote estrogen-independent growth of breast cancer cells, and increased levels of IGF-I and IRS-1 correlate with tumor progression and increasing tumor size. Insulin and E₂ stimulate growth of MCF7 cells, and their effect is synergistic (2). However, in MCF7 cells that express antisense IRS-1 RNA, insulin failed to stimulate growth, whereas the growth effects of E₂ alone or in combination with insulin were blunted (2). In addition, insulin and E₂ each up-regulated ER protein expression and binding capacity in MCF7 cells. Interestingly, most cells that expressed antisense IRS-1 RNA demonstrated higher basal ER protein levels and a higher ER binding capacity; however, in those cells, insulin failed to up-regulate ER expression and E₂ decreased ER protein levels (2). Finally, the effects of IGF-I were also examined in MCF7 cells; after treatment with IGF-I for just 3 h, a decrease in ER protein and mRNA expression was noted and was attributed to decreased ER gene transcription, with no change in ER binding affinity (60).

OTHER AGENTS

Several groups have examined the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on ERα expression. Initial studies demonstrated that upon treatment of MCF7 cells with TPA, the number of estrogen receptors diminished in spite of an increase in total cellular protein (23). The reduction in ER levels corresponded to the inhibition of cell growth. However, TPA-resistant RPh4 cells showed no change in ER number. Saceda et al. verified the TPA-induced reduction in ER protein and also observed a decrease in steady-state mRNA levels to 20% of control levels (50). In the presence of cycloheximide, TPA retained the ability to down-regulate ER mRNA, indicating that mRNA levels were not affected by protein synthesis. Importantly, 100 nM TPA had no effect on gene transcription but decreased the half-life of ER mRNA, suggesting that TPA causes posttranscriptional destabilization (50). The mechanism of ER regulation by TPA was further defined when the ability of the PKC inhibitor H-7 to block TPA-mediated effects on ER was shown (50). Mezerezein, a tumor promoter and activator of PKC, reduced ER mRNA by 80% (36). Ree et al. extended these findings by showing that the RNA synthesis inhibitor actinomycin D increased the ER mRNA half-life from 3 to 12 h and eliminated the TPA-induced decrease in ER mRNA levels (47). However, studies by Lee et al. contradicted earlier observations (36, 50). By using 10 nM TPA in contrast to 100 nM TPA, Lee et al. discovered that TPA caused a decrease in the ER gene transcription rate prior to the observed reduction in ER mRNA (36). Furthermore, they did not see an effect on ER mRNA stability. Thus, these studies conflict on whether TPA mediates ER mRNA transcriptionally or posttranscriptionally. The effects of TPA on ER have also been examined in leukemia cells (12). In THP-1 cells derived from a patient with acute monocytic leukemia, 50 ng of TPA/ml stimulated ER expression, whereas untreated cells were ER negative. Approximately 60 to 75% of TPA-treated cells stained positively for ER, with the majority of ER seen within the nucleus (12).

Various other agents have been evaluated for their effects on ER regulation. For example, in MCF7 cells, the heavy metal cadmium decreases the levels of both ER protein and mRNA while stimulating cell growth (21). In vitro nuclear run-on experiments demonstrated that cadmium induced a 60% inhibition of ER gene transcription. Arsenic compounds also modulate the expression of ER (6, 61). In MCF7 cells, arsenite caused a concentration-dependent decrease in both ER protein and mRNA by up to 90%. Arsenic trioxide, used in the treatment of patients with acute promyelocytic leukemia, significantly reduced the levels of ER expression in both MCF7 and T47D cells when given at low doses. At higher doses, this agent caused cell death. If cells were switched to medium free of arsenic trioxide, ER levels were completely restored.

CONCLUSIONS

Effectors of chromatin structure, hormones, growth factors, and a variety of other agents implicate multiple cellular systems in the control of ERα expression. The dramatic increase in information about these agents provides investigators with an enhanced conceptual framework for the role of estrogen biology in health and disease. The factors reviewed above serve as tools that can be utilized to further accelerate the pace of discovery in this area. Moreover, the diversity of ER regulatory agents reflects the biologic importance of precisely coordinating the timing and extent of ER expression and suggests that investigators working in a number of fields can contribute relevant molecular knowledge that may result in the clinical use of novel molecular determinants of ER expression.