Estrogen-mediated Regulation of Igf1 Transcription and Uterine Growth Involves Direct Binding of Estrogen Receptor to Estrogen-responsive Elements
ABSTRACT Estrogen enables uterine proliferation, which depends on synthesis of the IGF1 growth factor. This proliferation and IGF1 synthesis requires the estrogen receptor (ER), which binds directly to target DNA sequences (estrogen-responsive elements or EREs), or interacts with other transcription factors, such as AP1, to impact transcription. We observe neither uterine growth nor an increase in Igf1 transcript in a mouse with a DNA-binding mutated ER alpha (KIKO), indicating that both Igf1 regulation and uterine proliferation require the DNA binding function of the ER. We identified several potential EREs in the Igf1 gene, and chromatin immunoprecipitation analysis revealed ER alpha binding to these EREs in wild type but not KIKO chromatin. STAT5 is also reported to regulate Igf1; uterine Stat5a transcript is increased by estradiol (E(2)), but not in KIKO or alpha ERKO uteri, indicating ER alpha- and ERE-dependent regulation. ER alpha binds to a potential Stat5a ERE. We hypothesize that E(2) increases Stat5a transcript through ERE binding; that ER alpha, either alone or together with STAT5, then acts to increase Igf1 transcription; and that the resulting lack of IGF1 impairs KIKO uterine growth. Treatment with exogenous IGF1, alone or in combination with E(2), induces proliferation in wild type but not KIKO uteri, indicating that IGF1 replacement does not rescue the KIKO proliferative response. Together, these observations suggest in contrast to previous in vitro studies of IGF-1 regulation involving AP1 motifs that direct ER alpha-DNA interaction is required to increase Igf1 transcription. Additionally, full ER alpha function is needed to mediate other cellular signals of the growth factor for uterine growth.
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ABSTRACT: Inhibition of insulin-like growth factor I (IGF–I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2′-deoxycytidine and 5-propynyl-2′-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells.FEBS Journal 03/2014; · 3.99 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and disordered cognition. Women have a higher AD incidence than men, indicating that the declining estrogen levels during menopause may influence AD pathogenesis. However, the mechanism underlying estrogen's neuroprotective effect is not fully clarified and is complicated by the presence of several distinct estrogen receptor (ER) types and the identification of a growing number of ER splice variants. Thus, a deeper analysis of ERs could elucidate the role of estrogen in age-related cognitive changes. Intracellular calcium signaling cascades play a pivotal role in ERα neuroprotection against AD. The ERα-mediated inhibition of Death domain-associated protein (Daxx) translocation and the combination of membrane ERα and caveolin in caveolae may protect against AD. Moreover, the voltage-dependent anion channel (VDAC)/ERα association may be important for maintaining channel inactivation and may be relevant in neuronal preservation against Aβ injury. Additionally, ERα may prevent glutamate excitotoxic injury by Aβ through estrogen signaling mechanisms. ERα and IGF-IR co-activation may mediate neuroprotection, and many other growth factors and intracellular signaling responses triggered by ERα may also play important roles in this process. Furthermore, details regarding the genes and mRNA variants of ERα that are expressed in different parts of the human organs have been clarified recently. Therefore, here we review the literature to clarify the neuroprotective role of ERα. This review focuses on the potential mechanisms mediated by ERα in the intracellular signaling events in nervous system cells, thereby clarifying ERα-mediated protections against AD.Journal of Alzheimer's disease: JAD 08/2014; · 3.61 Impact Factor
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ABSTRACT: Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of the estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knockout (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Since adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned media (CM) from ERα-null adipocytes and media containing pure Lcn2 increased proliferation and migration of a sub-set of BrCA cells in culture. The proliferative and pro-migratory effects of ERα-deficient adipocyte CM on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. Lcn2-R (slc22a17) and 3-hydroxy butyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 12/2014; · 4.60 Impact Factor