Abstract17β‐Estradiol (E2), as the main circulating estrogen hormone, regulates many tissue and organ functions in physiology. The effects of E2 on cells are mediated by the transcription factors and estrogen receptor (ER)α and ERβ that are encoded by distinct genes. Localized at the peri‐membrane, mitochondria, and the nucleus of cells that are dependent on estrogen target tissues, the ERs share similar, as well as distinct, regulatory potentials. Different intracellular localizations of the ERs result in dynamically integrated and finely tuned E2 signaling cascades that orchestrate cellular growth, differentiation, and death. The deregulation of E2–ER signaling plays a critical role in the initiation and progression of target tissue malignancies. A better understanding of the complex regulatory mechanisms that underlie ER actions in response to E2 therefore holds a critical trajectory for the development of novel prognostic and therapeutic approaches with substantial impacts on the systemic management of target tissue diseases.

The effects of estrogens, particularly 17␤-estradiol (E2), are mediated by estrogen receptor ␣ (ER␣) and ER␤. Upon binding to E2, ERs homo-and heterodimerize when coexpressed. The ER dimer then regulates the transcription of target genes through estrogen responsive element (ERE)-dependent and -independent pathways that constitute genomic estrogen signaling. Although ER␣ and ER␤ have similar ERE and E2 binding properties, they display different transregulatory capacities in both ERE-dependent and -independent signaling pathways. It is therefore likely that the heterodimerization provides novel functions to ERs by combining distinct properties of the contributing partners. The elucidation of the role of the ER heterodimer is critical for the understanding of physiology and pathophysiology of E2 signaling. However, differentially determining target gene responses during cosynthesis of ER subtypes is difficult, since dimers formed are a heterogeneous population of homo-and heterodimers. To circumvent the pivotal dimerization step in ER action and hence produce a homogeneous ER heterodimer population, we utilized a genetic fusion strategy. We joined the cDNAs of ER␣ and/or ER␤ to produce single-chain ERs to simulate the ER homo-and heterodimers. The fusion ERs interacted with ERE and E2 in a manner similar to that observed with the ER dimers. The homofusion receptors mimicked the functions of the parent ER dimers in the ERE-dependent and -independent pathways in transfected mammalian cells, whereas heterofusion receptors emulated the transregulatory properties of the ER␣ dimer. These results suggest that ER␣ is the functionally dominant partner in the ER␣/␤ heterodimer.Estrogen hormones, particularly 17␤-estradiol (E2), exert their effects through a complex array of convergent and divergent signaling pathways that mediate genomic and nongenomic events, resulting in target tissue-specific responses (11, 31). The E2 information is conveyed by the transcription factors, estrogen receptor ␣ (ER␣) and ER␤ (11, 31), which are encoded by distinct genes and are expressed in different tissues as well as in the same tissue at various levels (11,31).Upon binding to E2, ER dimerizes and interacts with permutations of a palindromic DNA sequence separated by three nonspecific nucleotides: 5Ј-GGTCAnnnTGACC-3Ј, the consensus estrogen responsive element (ERE) (11,18,31). The E2-ER-ERE complex subsequently recruits coactivators/regulators to promote local chromatin remodeling and to bridge with general transcription factors for the initiation of transcription (11, 31). This pathway is called ERE-dependent ER signaling. The E2-ER complex also regulates gene expression through functional tethering to a transcription factor bound to its cognate regulatory element on DNA. This is the DNAdependent and ERE-independent signaling pathway (22, 36). Furthermore, E2 elicits effects through the membrane and cytoplasmic ERs (24, 39).ER␣ and ER␤ share high amino acid identity (96%) in their DNA-binding domains (DBDs) (11, 31), which is reflected in...

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Estrogen receptors (ER) alpha and beta are members of a superfamily of nuclear receptors and mediate estrogen [17beta-estradiol (E2)] signaling. ERbeta has considerably less transcription potency than ERalpha in heterologous expression systems that use E2 response elements (ERE) in tandem as the trans-acting unit. We show here that despite similar intracellular characteristics, ERbeta, in contrast to ERalpha, fails to induce gene transcription synergistically in response to E2 from tandem EREs. Moreover, our results indicate that ERalpha-specific partial agonistic activity of antagonists occurs additively. Although synergy contributes, it is not sufficient for differences in the transcription potencies between the ER subtypes. We demonstrate here that differences in the abilities of ERs to integrate activation functions through functional interactions between amino and carboxyl termini are critical for the transcriptional strength of ER subtypes.

Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) ␣ and ␤. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ER␣ through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ER␣ also modulates gene expression by interacting with transregulators bound to cognate DNA-regulatory elements, and this regulation is referred to as the ERE-independent signaling pathway. The relative importance of the ERE-independent pathway in E2-ER␣ signaling is unclear. To address this issue, we engineered an ERE-binding defective ER␣ mutant (ER␣ EBD ) by changing residues in an ␣-helix of the protein involved in DNA binding to render the receptor functional only through the ERE-independent signaling pathway. Using recombinant adenovirus-infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that E2-ER␣ EBD modulated the expression of a subset of ER␣-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ER␣ EBD did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ER␣. ER␣ EBD in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ER␣, on the other hand, effectively repressed growth in this cell line. Our findings suggest that genomic responses from the ERE-dependent signaling pathway are required for E2-ER␣ to induce alterations in cellular responses. 17␤-Estradiol (E2),5 as the main circulating estrogen hormone, plays critical roles in the physiology and pathophysiology of many tissues (1, 2). The effects of E2 are primarily mediated by estrogen receptor (ER) ␣ and ␤ (1, 2). ERs display functionally distinct structural features. The amino terminus of ER␣ contains a ligand-independent transactivation function. The central region is the DNA binding domain (DBD). The flexible hinge domain contains a nuclear localization signal and links the DBD domain to the multifunctional carboxyl-terminal ligand binding (LBD) domain. The LBD is involved in ligand binding, dimerization, and ligand-dependent transactivation function.Following synthesis, ER␣ dimerizes and translocates to the nucleus independent of E2 (3). Fractions of the ER␣ population also partition to the perimembrane, cytoplasm, and mitochondria (4). The binding of E2 to ER␣ leads to a major structural reorganization of the LBD that converts the inactive ER␣ to the functionally active form by generating surfaces that support protein-protein interactions (5). The integration of E2-ER␣ signaling generated from various cellular locations is thought to be critical for the regulation of responsive gene expression involved in cellular proliferation, differentiation, motility, and death (4, 6).One of the primary nuclear E2-ER␣ signaling events involves the interaction of E2-ER␣ with specific DNA sequences, known as estrogen-responsive e...

Estrogen hormone 17b-estradiol (E 2 ) is involved in the physiology and pathology of many tissues. E 2 information is conveyed by the transcription factors estrogen receptors (ER) a and b that mediate a complex array of nuclear and non-nuclear events. The interaction of ER with specific DNA sequences, estrogen-responsive elements (EREs), constitutes a critical nuclear signaling pathway. In addition, E 2 -ER regulates transcription through interactions with transfactors bound to their cognate regulatory elements on DNA, hence the ERE-independent signaling pathway. However, the relative importance of the EREindependent pathway in E 2 -ERb signaling is unclear. To address this issue, we engineered an ERE-binding defective ERb mutant (ERb EBD ) by changing critical residues in the DNA-binding domain required for ERE binding. Biochemical and functional studies revealed that ERb EBD signaled exclusively through the ERE-independent pathway. Using the adenovirus infected ER-negative cancer cell models, we found that although E 2 -ERb EBD regulated the expression of a number of genes identified by microarrays, it was ineffective in altering cellular proliferation, motility, and death in contrast to E 2 -ERb. Our results indicate that genomic responses from the ERE-independent pathway to E 2 -ERb are not sufficient to alter the cellular phenotype. These findings suggest that the ERE-dependent pathway is a required signaling route for E 2 -ERb to induce cellular responses.

Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre-carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.

The functions of 17beta-estradiol (E2) are mediated by estrogen receptor (ER) alpha and beta. ERs display similar DNA- and ligand-binding properties in vitro. However, ERbeta shows lower transcriptional activity than ERalpha from the estrogen response element (ERE)-dependent signaling. We predicted that distinct amino termini contribute to differences in transcription efficacies of ERs by affecting in situ ER-ERE interactions. We used chromatin immunoprecipitation and a novel in situ ERE competition assay, which is based on the ability of ER to compete for ERE binding with a designer activator that constitutively induces transcription from an ERE-driven reporter construct. Interference of activator-mediated transcription by unliganded or liganded ERs was taken as an indication of ER-ERE interaction. Results revealed that ERs interacted with ERE similarly in the absence of E2. However, E2 enhanced the ERE binding of ERalpha but not that of ERbeta. The removal of the amino terminus increased the ERbeta-ERE interaction independent of E2. The ERbeta amino terminus also prevented E2-mediated enhancement of the chimeric ERalpha-ERE interaction. Thus, the amino terminus of ERbeta impairs the binding of ERbeta to ERE. The abrogation of ligand-dependent activation function 2 of the amino-terminally truncated ERbeta resulted in the manifestation of E2 effect on ERbeta-ERE interaction. This implies that E2-mediated enhancement of ERbeta-ERE interaction is masked by the activation function 2, whereas the intact amino terminus is a dominant region that decreases the binding of ERbeta to ERE. Thus, ERbeta-ERE interaction is independent of E2 and is impaired by its amino terminus. These findings provide an additional explanation for differences between ERalpha and ERbeta functions that could differentially affect the physiology and pathophysiology of E2 signaling.