Extraembryonic heparin-binding epidermal growth factor-like growth factor deficiency compromises placentation in mice.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells.

During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2's effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Modulation of nuclear receptor activity by the F domain.

The F domain located at the C-terminus of proteins is one of the least conserved regions of the estrogen receptors alpha and beta, members of the nuclear hormone receptor superfamily. Indeed, many members of the superfamily lack the F domain. However, when present, removing the F domain entirely or mutating it alters transactivation, dimerization, and the responses to agonist and antagonist ligands. This review focuses on the functions of the F domain of the estrogen receptors, particularly in relation to other members of the superfamily.

The multifunctional estrogen receptor-alpha F domain.

The members of the nuclear receptor superfamily act as transcriptional regulatory factors and exhibit a multidomain structure characterized as domains A-E/F. This review focuses on a small, relatively understudied region at the extreme carboxy-terminus of the estrogen receptor (ER) alpha, the F domain. The F domain contributes to differences in the activity of ER alpha and beta subtypes; it is required for tamoxifen's agonist activity on an estrogen response element, and it modifies the receptor's interactions with coregulators including steroid receptor coactivator-1. The differences between the F domains of the ER alpha and beta subtypes and among the other members of the nuclear hormone receptor superfamily may offer opportunities for selective control of the activity of these proteins.

Reproductive factors, hormone use, estrogen receptor expression and risk of non small-cell lung cancer in women.

PURPOSE: Estrogen receptor (ER) expression in lung tumors suggests that estrogens may play a role in the development of lung cancer. We evaluated the role of hormone-related factors in determining risk of non-small-cell lung cancer (NSCLC) in women. We also evaluated whether risk factors were differentially associated with cytoplasmic ER-alpha and/or nuclear ER-beta expression-defined NSCLC in postmenopausal women. PATIENTS AND METHODS: Population-based participants included women aged 18 to 74 years diagnosed with NSCLC in metropolitan Detroit between November 1, 2001 and October 31, 2005. Population-based controls were identified through random digit dialing, matched to patient cases on race and 5-year age group. Interview data were analyzed for 488 patient cases (241 with tumor ER results) and 498 controls. RESULTS: Increased duration of hormone replacement therapy (HRT) use in quartiles was associated with decreased risk of NSCLC in postmenopausal women (odds ratio = 0.88; 95% CI, 0.78 to 1.00; P = .04), adjusting for age, race, pack-years, education, family history of lung cancer, current body mass index, years exposed to second-hand smoke in the workplace, and obstructive lung disease history. Among postmenopausal women, ever using HRT, increasing HRT duration of use in quartiles, and increasing quartiles of estrogen use were significant predictors of reduced risk of NSCLC characterized as ER-alpha and/or ER-beta positive. None of the hormone-related variables were associated with nuclear ER-alpha- or ER-beta-negative NSCLC. CONCLUSION: These findings suggest that postmenopausal hormone exposures are associated with reduced risk of ER-alpha- and ER-beta-expressing NSCLC. Understanding tumor characteristics may direct development of targeted treatment for this disease.

Identification of regions within the F domain of the human estrogen receptor alpha that are important for modulating transactivation and protein-protein interactions.

The estrogen receptor (ER)alpha is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERalpha modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ERalpha (hERalpha) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins ("monobodies") that specifically bind to ERalpha/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579-584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a beta-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERalpha ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERalpha selectively modulate its activity and its interactions with other proteins.

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen.

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.

Understanding the human estrogen receptor-alpha using targeted mutagenesis.

The estrogen receptor-alpha is a wonderfully complex protein important in normal biology, breast cancer, and as a target for anti-cancer agents. We are using the available structures of the hERalpha as well as secondary structure predictions to guide site-directed mutagenesis in order to test the importance of specific interactions and regions in the ligand-regulated activity of the protein. In one area of interest, we are investigating the role of the F domain in the ligand-stimulated activity of the hERalpha. Results from our laboratory and others suggest that the F domain modulates the activity of the hERalpha. In order to better understand the role of the F domain in the hERalpha, we have constructed mutants within this region. Mutations within a predicted alpha-helical region alter the response of the ER to estradiol (E2), eliminate or impair the agonist activity of 4-hydroxytamoxifen (4-OHT), and alter the ability of E2 to overcome 4-OHT's antagonist activity. Deleting the F domain increases the affinity of the receptor for E2; by contrast, mutating a residue in the middle of the predicted helix to a proline does not alter the affinity for E2, but does change the binding mechanism from a positive cooperative to a noncooperative interaction. These and other results show the F domain exhibits substantial functional complexity, and support the idea that this domain modulates the activity of the hERalpha. In a second area of interest, we are investigating the role of hydrophobic and hydrogen-bonding interactions at the start of helix 12 in the activity of the hERalpha. Leucine-536 (L536) has been proposed to participate in hydrophobic interactions that form part of a capping motif stabilizing the start of helix 12. When mutated, the resulting receptors exhibit a reduced response, or even an inverted response, to E2 and 4-OHT on both ERE-driven and AP-1-driven promoters. Interestingly, these mutated receptors also exhibit altered interactions with probes that recognize the agonist-bound and 4-OHT-bound conformations of the ERalpha. Thus, L536 couples the binding of ligand with the conformation of the receptor. Overall, these results show that combining structure-based hypotheses with functional tests of the ER's activity can identify regions and interactions that are important in the ligand-stimulated activity of the protein.

Targeted mutation of key residues at the start of helix 12 in the hERalpha ligand-binding domain identifies the role of hydrogen-bonding and hydrophobic interactions in the activity of the protein.

Estradiol (E(2)) and tamoxifen exert their effects through two members of the nuclear receptor superfamily, estrogen receptor (ER)-alpha and -beta. We want to identify the key interactions linking ligand-binding and activity of the ERalpha. Asp-351 and Leu-536 participate in hydrogen bond (Asp-351) and hydrophobic (Leu-536) interactions at the start of helix 12 in the ligand-binding domain (LBD) of the ERalpha. Mutations at each position alter ER activity, but we do not know which is more important. We mutated these residues in combination and individually and assessed the activity of the mutated ERs in the absence and presence of E(2) and 4-OHT on an ERE-driven and an AP-1-driven promoter, as well as their ability to interact with coregulators. On an ERE-driven promoter, the residue at position 351 determined whether E(2) stimulated or reduced the activity of the ER, as well as the level of activity in the presence of 4-OHT. Surprisingly, mutation of both residues generally did not produce cumulative deleterious effects, and they exerted counterbalancing effects on the basal activity on both promoters. Our results identify the contributions of specific interactions to the activity of the hERalpha, and support the concept that this region couples ligand-binding with ER activity.

From ligand structure to biological activity: modified estratrienes and their estrogenic and antiestrogenic effects in MCF-7 cells.

A variety of compounds, including the selective estrogen receptor (ER) modulators tamoxifen and raloxifene, phytoestrogens such as genistein, and xenoestrogens such as bisphenol, bind to the estrogen receptor and elicit biological responses. Structural studies have linked the altered activity of compounds such as 4-hydroxytamoxifen, raloxifene, genistein, and tetrahydrochrysene, which have substantially different structures from estradiol (E2), to differences in the positioning of the critical "helix 12" within the ligand-binding domain (LBD) of the ER-ligand complex. However, subtle permutations of the E2 molecule would also be expected to modulate the pattern of responses within a cell. Forty-two ligands were constructed by the addition or relocation of double bonds, hydroxyl, keto, amino, and nitro substituents throughout the estra-l,3,5(10)-triene (estratriene) ring system. In this review, we summarize the effects of subtle changes in the estratriene molecule on the ability of the receptor complex to stimulate the growth of MCF-7 cells, or affect the expression of four estrogen-regulated genes (progesterone receptor, pS2 protein, cathepsin D, and tissue plasminogen activator), as well as undergo nuclear processing and downregulate ERalpha mRNA. The affinity of these ligands for, and mechanism of their binding with, the ERalpha have been measured, along with their effect on the conformation of the ER-ERE complex. In particular, two A-ring isomers of E2, 2- and 4-hydroxyestratriene-17beta-ol, display gene selective activity within MCF-7 cells which is dependent on complex endogenous promoters, an intact AF-2 and is sensitive to the level of SRC-1. Both of these A-ring isomers function as antiestrogens. Molecular modeling of these two A-ring isomers complexed with the ER ligand-binding domain supports the idea that the conformation of the LBD is affected by subtle changes in the estratriene structure.

Mutation of Leu-536 in human estrogen receptor-alpha alters the coupling between ligand binding, transcription activation, and receptor conformation.

The estrogen receptor (ER), of which there are two forms, ERalpha and ERbeta, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ERalpha ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ERalpha, as well as in the ability of antagonists to block the activity of ERalpha. We found that certain mutations of Leu-536 increased the ligand-independent activity of ERalpha although greatly reducing or eliminating the agonist activity of 17beta-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ERalpha to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2. Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ERalpha and increased the reactivity of the raloxifene- or ICI 182,780-bound ERalpha, with probes that recognize the 4OHT-bound ERalpha conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ERalpha.

Mutations targeted to a predicted helix in the extreme carboxyl-terminal region of the human estrogen receptor-alpha alter its response to estradiol and 4-hydroxytamoxifen.

The human estrogen receptor-alpha, a member of the nuclear receptor superfamily, is a ligand-regulated transcriptional modulator. Because comparatively little is known about the extreme carboxyl-terminal region of the estrogen receptor (F domain), we used secondary structure prediction to design mutations that delete the F domain (S554stop), disrupt a possible turn (G556L/G557L), and alter a predicted helix (S559A/E562A, Q565P), and we evaluated the effects of these mutations on hormone binding and transcription activation in response to estradiol and the mixed agonist/antagonist 4-hydroxytamoxifen. Mutations that deleted the F domain (S554stop) or targeted the predicted helix (S559A/E562A, Q565P) greatly reduced or eliminated the agonist activity of 4-hydroxytamoxifen. Deleting the F domain increased the affinity of the receptor for estradiol and decreased the antagonist activity of 4-hydroxytamoxifen. The Q565P mutant exhibited a non-cooperative hormone-binding mechanism, as well as an impaired response to estradiol and increased antagonist activity of 4-hydroxytamoxifen. Our results show that mutations in the F domain alter not only the response to estradiol, the affinity for hormone, and the interaction between receptor subunits but can uncouple the agonist and antagonist activities of 4-hydroxytamoxifen. These results suggest that the F domain modulates the activity of the estrogen receptor-alpha by multiple mechanisms.