Estrogen-dependent and -independent estrogen receptor-alpha signaling separately regulate male fertility.
ABSTRACT: Estrogen receptor-alpha (ERalpha) plays a critical role in male reproductive tract development and fertility. To determine whether estrogen-dependent and -independent ERalpha mechanisms are involved in male fertility, we examined male estrogen nonresponsive ERalpha knock-in mice. These animals have a point mutation (G525L) in the ligand-binding domain of ERalpha that significantly reduces interaction with, and response to, endogenous estrogens but does not affect growth factor activation of ligand-independent ERalpha pathways. Surprisingly, we found that ligand-independent ERalpha signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption. In contrast, estrogen-dependent ERalpha signaling is required for germ cell viability, most likely through support of Sertoli cell function. By treating estrogen nonresponsive ERalpha knock-in (ENERKI) mice with the ERalpha selective synthetic agonist propyl pyrazole triol, which is able to bind and activate G525L ERalpha in vivo, we discovered male fertility required neonatal estrogen-mediated ERalpha signaling. Thus, our work indicates both estrogen-dependent and -independent pathways play separable roles in male murine reproductive tract development and that the role of ERalpha in human infertility should be examined more closely.
Project description:Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.
Project description:Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERalpha also enhances insulin synthesis in cultured islets. Whether ERalpha stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERalpha knockout mouse (PERalpha KO(-/-)) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and beta cells. Whereas 17beta-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERalpha lox/lox mice, these E2 effects are abolished in PERalpha KO(-/-) mice. We find that E2-activated ERalpha increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERalpha eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERalpha interacts with the tyrosine kinase Src, which activates extracellular signal-regulated kinases(1/2), to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERalpha in beta cells as a regulator of insulin synthesis in vivo.
Project description:The estrogen receptor alpha (ERalpha) is activated as a transcription factor by both estrogen and a large variety of other extracellular signals. The mechanisms of this ligand-independent activation, notably by cAMP signaling, are still largely unknown. We now close the gap in the signaling pathway between cAMP and ERalpha. Whereas the direct phosphorylation of ERalpha by the cAMP-activated protein kinase A (PKA) is dispensable, the phosphorylation of the coactivator-associated arginine methyltransferase 1 (CARM1) by PKA at a single serine is necessary and sufficient for direct binding to the unliganded hormone-binding domain (HBD) of ERalpha, and the interaction is necessary for cAMP activation of ERalpha. Sustained PKA activity promoting a constitutive interaction may contribute to tamoxifen resistance of breast tumors. Binding and activation involve a novel regulatory groove of the ERalpha HBD. As a result, depending on the activating signal, ERalpha recruits different coactivator complexes to regulate alternate sets of target genes.
Project description:Treatment with either estradiol or an estrogen receptor (ER)alpha ligand has been shown to be both antiinflammatory and neuroprotective in a variety of neurological disease models, but whether neuroprotective effects could be observed in the absence of an antiinflammatory effect has remained unknown. Here, we have contrasted effects of treatment with an ERalpha vs. an ERbeta ligand in experimental autoimmune encephalomyelitis, the multiple sclerosis model with a known pathogenic role for both inflammation and neurodegeneration. Clinically, ERalpha ligand treatment abrogated disease at the onset and throughout the disease course. In contrast, ERbeta ligand treatment had no effect at disease onset but promoted recovery during the chronic phase of the disease. ERalpha ligand treatment was antiinflammatory in the systemic immune system, whereas ERbeta ligand treatment was not. Also, ERalpha ligand treatment reduced CNS inflammation, whereas ERbeta ligand treatment did not. Interestingly, treatment with either the ERalpha or the ERbeta ligand was neuroprotective, as evidenced by reduced demyelination and preservation of axon numbers in white matter, as well as decreased neuronal abnormalities in gray matter. Thus, by using the ERbeta selective ligand, we have dissociated the antiinflammatory effect from the neuroprotective effect of estrogen treatment and have shown that neuroprotective effects of estrogen treatment do not necessarily depend on antiinflammatory properties. Together, these findings suggest that ERbeta ligand treatment should be explored as a potential neuroprotective strategy in multiple sclerosis and other neurodegenerative diseases, particularly because estrogen-related toxicities such as breast and uterine cancer are mediated through ERalpha.
Project description:Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.
Project description:Conjugated equine estrogens (CEEs) are routinely used for hormone replacement therapy (HRT), making it important to understand the activities of individual estrogenic components. Although 17beta-estradiol (17beta-E2), the most potent estrogen in CEE, has been extensively characterized, the actions of nine additional less potent estrogens are not well understood. Structural differences between CEEs and 17beta-E2 result in altered interactions with the two estrogen receptors (ERalpha and ERbeta) and different biological activities. To better understand these interactions, we have determined the crystal structure of the CEE analog, 17beta-methyl-17alpha-dihydroequilenin (NCI 122), in complex with the ERalpha ligand-binding domain and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator. NCI 122 has chemical properties, including an unsaturated B-ring and 17alpha-hydroxyl group, which are shared with some of the estrogens found in CEEs. Structural analysis of the NCI 122-ERalpha LBD-GRIP1 complex, combined with biochemical and cell-based comparisons of CEE components, suggests that factors such as decreased ligand flexibility, decreased ligand hydrophobicity and loss of a hydrogen bond between the 17-hydroxyl group and His524, contribute significantly to the reduced potency of CEEs on ERalpha.
Project description:Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERalpha) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERalpha has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERalpha represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERalpha gene, ESR1. Upon estrogen treatment, ERalpha is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERalpha-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERalpha is identified. These data support a model of repression wherein actions of ERalpha and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.
Project description:Transcriptional repression and activation by nuclear receptors (NRs) are brought about by coregulator complexes. These complexes modify the chromatin environment of target genes and affect the activity of the basal transcription machinery. We have previously implicated the yeast ADA3 protein in transcriptional activation by estrogen and retinoid X receptors in yeast and mammalian cells. Here we report the cloning of the mouse homolog of ADA3 and its characterization with respect to the estrogen receptor alpha (ERalpha) function. Mouse mADA3 is 23% identical and 47% similar to yeast yADA3, and mADA3 in contrast to yADA3 does not interact with NRs directly even though it contains two LxxLL NR boxes. However, the ADA3-containing TBP-free-TAF-containing complex (TFTC) can interact with ERalpha in a ligand-independent manner, indicating that other subunits of the complex are sufficient to mediate interaction with NRs.
Project description:Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) alpha and beta. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ERalpha through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ERalpha 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-ERalpha signaling is unclear. To address this issue, we engineered an ERE-binding defective ERalpha mutant (ERalpha(EBD)) by changing residues in an alpha-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-ERalpha(EBD) modulated the expression of a subset of ERalpha-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ERalpha(EBD) did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ERalpha.ERalpha(EBD) in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ERalpha, 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-ERalpha to induce alterations in cellular responses.
Project description:Estrogen is atheroprotective and a high-affinity ligand for both known estrogen receptors, ERalpha and ERbeta. However, the role of the ERalpha in early-stage atherosclerosis has not been directly investigated and is incompletely understood. ERalpha-deficient (ERalpha-/-) and wild-type (ERalpha+/+) female mice consuming an atherogenic diet were studied concurrent with estrogen replacement to distinguish the actions of 17beta-estradiol (E(2)) from those of ERalpha on the development of early atherosclerotic lesions. Mice were ovariectomized and implanted with subcutaneous slow-release pellets designed to deliver 6 or 8 mug/day of exogenous 17beta-estradiol (E(2)) for a period of up to 4 months. Ovariectomized mice (OVX) with placebo pellets (E(2)-deficient controls) were compared to mice with endogenous E(2) (intact ovaries) and exogenous E(2). Aortas were analyzed for lesion area, number, and distribution. Lipid and hormone levels were also determined. Compared to OVX, early lesion development was significantly (p < 0.001) attenuated by E(2) with 55-64% reduction in lesion area by endogenous E(2) and >90% reduction by exogenous E(2). Compared to OVX, a decline in lesion number (2- to 4-fold) and lesser predilection (~4-fold) of lesion formation in the proximal aorta also occurred with E(2). Lesion size, development, number, and distribution inversely correlated with circulating plasma E(2) levels. However, atheroprotection was independent of ERalpha status, and E(2) athero-protection in both genotypes was not explained by changes in plasma lipid levels (total cholesterol, triglyceride, and high-density lipoprotein cholesterol). The ERalpha is not essential for endogenous/exogenous E(2)-mediated protection against early-stage atherosclerosis. These observations have potentially significant implications for understanding the molecular and cellular mechanisms and timing of estrogen action in different estrogen receptor (ER) deletion murine models of atherosclerosis, as well as implications to human studies of ER polymorphisms and lipid metabolism. Our findings may contribute to future improved clinical decision-making concerning the use of hormone therapy.