Jeffrey Weiss

Estrogen Receptor Binding to DNA Is Not Required for Its Activity through the Nonclassical AP1 Pathway

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERα. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.

Nonclassical estrogen receptor α signaling mediates negative feedback in the female mouse reproductive axis

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) α but not ERβ knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERα. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERα signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERα (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERα-null (−/−) background, we examine the ability of ERE-independent ERα signaling pathways to convey estrogen feedback regulation of the female hypothalamic–pituitary axis in vivo. ERα−/AA exhibited 69.9% lower serum LH levels compared with ERα−/− mice. Additionally, like wild type, ERα−/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERα−/AA mice. However, unlike wild type, both ERα−/AA and ERα−/− mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERα signaling is sufficient to convey a major portion of estrogens negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERα signaling.

Sox3 Is Required for Gonadal Function, but Not Sex Determination, in Males and Females

ABSTRACT Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (−/−) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (−/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

Regulation of Kiss1 and Dynorphin Gene Expression in the Murine Brain by Classical and Nonclassical Estrogen Receptor Pathways

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17β (E2), which acts via the estrogen receptor α isoform (ERα) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E2 inhibits the expression of Kiss1 mRNA. However, E2 induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E2 is unknown. ERα signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E2 on Kiss1 and Dyn expression, we studied the effects of E2 on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ERα signaling pathways. We found that stimulation of Kiss1 expression by E2 in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E2 in the Arc involved ERE-independent mechanisms. Thus, distinct ERα signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E2 can act within the same neuron through divergent ERα signaling pathways to regulate different neurotransmitter genes.

Dax1 regulates testis cord organization during gonadal differentiation.

Mutations of the DAX1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Targeted deletion of Dax1 in mice also reveals primary testicular dysgenesis, which is manifest by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells. Because Dax1 is expressed early in gonadal development, and because Sertoli and Leydig cells are located ectopically in the adult, we hypothesized that these testis abnormalities are the result of an early defect in testis development. In Dax1-/Y males, the gonad develops normally until 12.5 dpc. However, by 13.5 dpc, the testis cords are disorganized and incompletely formed in Dax1-deficient mice. The number of germ and Sertoli cells is unchanged, and the expression of Sertoli-specific markers appears to be normal. However, the number of peritubular myoid cells, which normally surround the testis cords, is reduced. BrdU labeling of peritubular myoid cells is low, consistent with decreased proliferation. The basal lamina produced by peritubular myoid and Sertoli cells is disrupted, leading to open and incompletely formed testis cords. Leydig cells, which normally reside in the peritubular space and extend from the coelomic surface to the dorsal surface of the gonad, are restricted to the coelomic surface of Dax1-deficient testis. We conclude that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. The developmental abnormalities in the Dax1-deficient testis lay the foundation for gonadal dysgenesis and infertility in adult mice and, potentially in humans with DAX1 mutations.

Genetic rescue of nonclassical ERα signaling normalizes energy balance in obese Erα-null mutant mice

In addition to its role in reproduction, estradiol-17β is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα-/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα-/- mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17β on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women.

Estrogen-induced Proliferation of Uterine Epithelial Cells Is Independent of Estrogen Receptor α Binding to Classical Estrogen Response Elements

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERα, a mutant ERα (E207A/G208A) that selectively lacks ERE binding, or ERα null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17β-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERα signaling. This proliferative response was confirmed by 5-bromo-2′-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathwayresponsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERα (E207A/G208A) mutant. These results indicate that nonclassical ERα signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERα plays a critical physiologic role in the uterine response to estrogen.

Inherited disorders of the gonadotropin hormones

Pulsatile GnRH acts at the GnRH receptor on gonadotropes to stimulate gonadotropin gene expression, hormone synthesis and secretion. The pituitary gonadotropins, LH and FSH, stimulate steroid production and gametogenesis in males and in females. Gonadotropin production thus requires the normal development and function of hypothalamic GnRH-producing neurons and pituitary gonadotrope cells. Genes involved in gonadotrope development and/or gene expression include SF1, DAX1, KAL, GNRHR, PC1, HESX1, LHX3, PROP1, LH beta, and FSH beta. Given the complex control of gonadotropin biosynthesis and secretion, it is not surprising that genetic abnormalities have been identified at several of these steps. Some of the mutations that will be reviewed include: (1) SF1 and DAX1-orphan nuclear receptors that are expressed at multiple levels throughout the reproductive axis; (2) KAL-X-linked Kallmann syndrome, where there is abnormal development of hypothalamic GnRH-producing neurons; (3) PC1-causing abnormal processing of GnRH and GNRHR mutations that impair action at the GnRH receptor; (4) HESX1, LHX3, PROP1-abnormal development/function of the gonadotrope cell lineage; (5) LH beta and FSH beta-mutations in the gonadotropin genes that cause structural abnormalities in the hormones. Although all of these gene defects lead to gonadotropin deficiency, each disorder is associated with unique phenotypic or hormonal features. Characterization of the molecular basis of gonadotropin deficiency is useful for directing therapy and for genetic counseling. Identification of these mutations also provides insight into the pathways that govern reproduction.

Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development

Mutations of orphan nuclear receptors SF1 and DAX1 each cause adrenal insufficiency and gonadal dysgenesis in humans, although the pathological features are distinct. Because Dax1 antagonizes Sf1-mediated transcription in vitro, we hypothesized that Dax1 deficiency would compensate for allelic loss of Sf1. In studies of the developing testis, expression of the fetal Leydig cell markers Cyp17 and Cyp11a1 was reduced in heterozygous Sf1-deficient mice at E13.5, consistent with dose-dependent effects of Sf1. In Sf1/Dax1 (Sf1 heterozygous and Dax1-deleted) double mutant gonads, the expression of these genes was unexpectedly reduced further, indicating that loss of Dax1 did not compensate for reduced Sf1 activity. The Sertoli cell product Dhh was reduced in Sf1 heterozygotes at E11.5, and it was undetectable in Sf1/Dax1 double mutants, indicating that Sf1 and Dax1 function cooperatively to induce Dhh expression. Similarly, Amh expression was reduced in both Sf1 and Dax1 single mutants at E11.5, and it was not rescued by the Sf1/Dax1 double mutant. By contrast, Sox9 was expressed in single and in double mutants, suggesting that various Sertoli cell genes are differentially sensitive to Sf1 and Dax1 function. Reduced expression of Dhh and Amh was transient, and was largely restored by E12.5. Similarly, there was recovery of fetal Leydig cell markers by E14.5, indicating that loss of Sf1/Dax1 delays but does not preclude fetal Leydig cell development. Thus, although Sf1 and Dax1 function as transcriptional antagonists for many target genes in vitro, they act independently or cooperatively in vivo during male gonadal development.

A murine model of autosomal dominant neurohypophyseal diabetes insipidus reveals progressive loss of vasopressin-producing neurons

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the arginine vasopressin (AVP) precursor. The pathogenesis of FNDI is proposed to involve mutant protein-induced loss of AVP-producing neurons. We established murine knock-in models of two different naturally occurring human mutations that cause FNDI. A mutation in the AVP signal sequence [A(-1)T] is associated with a relatively mild phenotype or delayed presentation in humans. This mutation caused no apparent phenotype in mice. In contrast, heterozygous mice expressing a mutation that truncates the AVP precursor (C67X) exhibited polyuria and polydipsia by 2 months of age and these features of DI progressively worsened with age. Studies of the paraventricular and supraoptic nuclei revealed induction of the chaperone protein BiP and progressive loss of AVP-producing neurons relative to oxytocin-producing neurons. In addition, Avp gene products were not detected in the neuronal projections, suggesting retention of WT and mutant AVP precursors within the cell bodies. In summary, this murine model of FNDI recapitulates many features of the human disorder and demonstrates that expression of the mutant AVP precursor leads to progressive neuronal cell loss.