Heather A. Harris

The ligand binding profiles of estrogen receptors α and β are species dependent

Abstract Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-α and ER-β. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-α and ER-β. Certain compounds show species dependent binding preferences for ER-α or ER-β, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.

Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway.

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.

Activity of three selective estrogen receptor modulators on hormone-dependent responses in the mouse uterus and mammary gland.

Selective estrogen receptor modulators (SERMs) have the unique potential to provide estrogenic effects in the skeletal and cardiovascular system, while minimizing/eliminating side effects on reproductive organs. However, despite the unifying characteristic of mixed estrogen receptor (ER) agonist/antagonist activity, compounds within this class are not interchangeable. In order to define and compare the effects of SERMs on different hormone-responsive tissues, we evaluated effects of bazedoxifene acetate (BZA), lasofoxifene (LAS) and raloxifene (RAL) in the mammary gland and uterus of the ovariectomized mouse. Endpoints measured included those regulated by estradiol alone (uterine wet weight, uterine G protein-coupled receptor 105 (GPR105) mRNA expression and mammary gland indoleamine-pyrrole 2,3 dioxygenase (INDO) mRNA expression) as well as others that required the combination of estradiol and progesterone (uterine serine protease inhibitor Kazal type 3 (Spink3) mRNA expression, mammary gland morphology and mammary gland defensin beta1 (Defbeta1) mRNA expression). The three SERMs tested had variable agonist and antagonist activity on these endpoints. In the uterus, the SERMs were mixed agonists/antagonists on estradiol-induced wet weight increase, whereas all three SERMs were estrogen receptor antagonists on GPR105 mRNA expression. However, in the presence of progesterone, BZA and RAL were agonists on Spink3 expression, while LAS was primarily an antagonist. In the mammary gland, BZA and RAL were predominantly agonists on the endpoint of mammary morphology and all three SERMs were clear agonists on Defbeta1 mRNA expression, an E+P-dependent marker. Finally, LAS and RAL had mixed agonist/antagonist activity on INDO mRNA expression, while BZA had only antagonist activity. These results demonstrate that compounds with small structural differences can elicit distinct biological responses, and that in general, SERMs tended to behave more as antagonists on endpoints requiring estrogen alone and agonists on endpoints requiring the combination of estrogen and progesterone.

Constrained phytoestrogens and analogues as ERβ selective ligands

A new series of ERbeta (ERbeta) selective ligands has been prepared. One of the compounds 6, structurally related to the phytoestrogen apigenin 4, displays a binding preference for ERbeta over ERalpha of over 40-fold. In addition to its binding selectivity, 6 was able to potently induce metallothionein (an ERbeta specific response in human SAOS-2 cells) while demonstrating low potency in an ERalpha dependant ERE-tk luciferase assay in MCF-7 cells. Such receptor and cell selectivity could make 6 a useful molecular probe for better understanding the role of ERbeta in mammalian physiology.

Modulation of responses to stress by estradiol benzoate and selective estrogen receptor agonists

Previously, pretreatment with estradiol benzoate (EB) was found to modulate the response of hypothalamic-pituitary-adrenal (HPA) axis and gene expression in several catecholaminergic neuronal locations in ovariectomized (OVX) rats exposed to single immobilization stress (IMO). Here, we investigated the role of estrogen receptor (ER) subtypes, using selective agonists for ERalpha (propyl pyrazole triol, PPT) or ERbeta (WAY-200070) in two major central noradrenergic systems and the HPA axis after exposure to single and repeated IMO. OVX female rats received 21 daily injections of either EB (25 mug/kg), PPT (10 mg/kg), WAY-200070 (10 mg/kg), or vehicle. Injections of EB and PPT, but not WAY-200070, elicited reduced body weight and increased uterine weight, showing their selectivity. Both EB and PPT increased corticosterone levels about two- to threefold, but prevented any further rise with either single or repeated IMO, indicating an ERalpha (ESR1)-, but not ERbeta (ESR2)-, mediated mechanism. In the locus coeruleus (LC), the rise in dopamine-beta-hydroxylase (Dbh) mRNA with both stress paradigms was abrogated in EB- or PPT-injected animals. However, WAY-200070 blocked the response of DBH mRNA to single IMO but not to repeated IMO. In the nucleus of the solitary tract (NTS), the rise in tyrosine hydroxylase and DBH mRNAs with both IMOs was absent, or greatly attenuated, in EB- or PPT-treated rats. In most cases, WAY-200070 inhibited the response to single IMO but not to repeated IMO. The results demonstrate that pretreatment with estradiol, or ER-selective agonists, modulates the stress-triggered induction of gene expression of norepinephrine biosynthetic enzymes in LC and NTS, with ER selectivity depending on duration of the stress.

Development of a mouse model of mammary gland versus uterus tissue selectivity using estrogen- and progesterone-regulated gene markers.

We have identified mRNA markers of estradiol and progesterone action in the mouse mammary gland and uterus to establish an in vivo model for the evaluation of novel and potentially tissue selective estrogens and progestins. Gene chip analysis of mRNA from ovariectomized (OVX) mice treated with vehicle (V), 17beta-estradiol (E2), progesterone (P) or E2+P for 7 days identified defensinbeta1 (Defbeta1) and indoleamine-pyrrole 2,3 dioxygenase (INDO) as markers of E2 and P action in the mammary gland, and serine protease inhibitor, Kazal type 3 (Spink3) and G protein-coupled receptor 105 (GPR105) as markers in the uterus. Defbeta1 and Spink3 are both upregulated by E2+P, whereas INDO and GPR105 have a complementary profile of upregulation by E2 alone and suppression of the E2 effect by P. Quantitative RT-PCR analysis of mammary gland markers was concordant with histological changes. Using this model, medroxyprogesterone acetate (MPA) and tanaproget (TNPR), a novel nonsteroidal progesterone receptor agonist, were evaluated and found to have no marked tissue selectivity relative to progesterone. In addition, the ERalpha selective ligand propyl pyrazole triol (PPT) and the ERbeta selective ligands ERB-041 and WAY-202196 were evaluated on the mammary gland endpoints of histology and Defbeta1 mRNA expression, and showed that ERalpha stimulation is necessary and sufficient for eliciting estradiol-mediated changes in the mammary gland.

Estrogen Receptor β Agonism Increases Survival in Experimentally Induced Sepsis and Ameliorates the Genomic Sepsis Signature: A Pharmacogenomic Study

BACKGROUND Nonsteroidal agonists have been developed that selectively bind to and activate estrogen receptor beta (ERbeta) rather than estrogen receptor alpha (ERalpha). ERbeta is expressed equally in both male and female mammals in multiple extragonadal tissues. Work reported elsewhere has demonstrated that ERbeta agonists have beneficial effects in multiple (but not all) models of inflammatory diseases and also increase survival in experimentally induced sepsis. METHODS In these experiments, ERbeta agonists (ERB-041 or WAY-202196) were compared with vehicle control in the murine cecal ligation and puncture (CLP) model and in the pneumococcal pneumonia model of sepsis. The effect of WAY-202196 on the gene expression profile in the CLP model was further studied by transcriptome analysis of lung and small intestine tissue samples. RESULTS ERbeta agonists provided a significant survival benefit in both experimental models of bacterial sepsis. This survival advantage was accompanied by reduced histologic evidence of tissue damage, reduced transcription of multiple proinflammatory proteins by transcriptome analysis and was not associated with increased bacterial outgrowth. CONCLUSIONS ERbeta agonist administration provided a survival advantage in septic animals and appears to be a promising therapeutic modality in sepsis.

A Mouse Model of Androgenetic Alopecia

Androgenetic alopecia (AGA), commonly known as male pattern baldness, is a form of hair loss that occurs in both males and females. Although the exact cause of AGA is not known, it is associated with genetic predisposition through traits related to androgen synthesis/metabolism and androgen signaling mediated by the androgen receptor (AR). Current therapies for AGA show limited efficacy and are often associated with undesirable side effects. A major hurdle to developing new therapies for AGA is the lack of small animal models to support drug discovery research. Here, we report the first rodent model of AGA. Previous work demonstrating that the interaction between androgen-bound AR and beta-catenin can inhibit Wnt signaling led us to test the hypothesis that expression of AR in hair follicle cells could interfere with hair growth in an androgen-dependent manner. Transgenic mice overexpressing human AR in the skin under control of the keratin 5 promoter were generated. Keratin 5-human AR transgenic mice exposed to high levels of 5alpha-dihydrotestosterone showed delayed hair regeneration, mimicking the AGA scalp. This effect is AR mediated, because treatment with the AR antagonist hydroxyflutamide inhibited the effect of dihydrotestosterone on hair growth. These results support the hypothesis that androgen-mediated hair loss is AR dependent and suggest that AR and beta-catenin mediate this effect. These mice can now be used to test new therapeutic agents for the treatment of AGA, accelerating the drug discovery process.

Regulation of Metallothionein II Messenger Ribonucleic Acid Measures Exogenous Estrogen Receptor-β Activity in SAOS-2 and LNCaPLN3 Cells

Estrogen receptor-β (ERβ) is a recently discovered member of the steroid hormone superfamily. Because its distribution is distinct from that of the classical estrogen receptor, and it is expressed in several nonclassical estrogen target tissues (e.g. prostate and bladder), its role in mediating the action of estrogen is unclear. One approach to elucidating the function of this receptor is to identify genes that it regulates. Using differential display, we profiled the messenger RNAs regulated by 17β-estradiol in SAOS-2 and LNCaPLN3 cells overexpressing ERβ. Follow-up studies used cells expressing either ERα or ERβ. One gene, metallothionein II, was regulated by both receptor subtypes in LNCaPLN3 cells, but only by ERβ in SAOS-2 cells. Because cycloheximide blocks this response, induction is probably mediated through regulation of at least one other protein. Identification of endogenous genes that are regulated differentially by ERα and ERβ will be valuable tools in elucidating the function of ERβ and the ...

Expression of meltrin‐α mRNA is not restricted to fusagenic cells

Meltrin‐α is a myoblast gene product reported to be required for cell fusion [Yagami‐Hiromasa et al. (1995): Nature 377:652–656]. Because Northern blots revealed expression only in muscle and bone, the suggestion was made that meltrin‐α is expressed exclusively by fusagenic cells in these tissues (myoblast and osteoclast). We studied expression of meltrin‐α mRNA in a panel of tissues and cell lines using the polymerase chain reaction and found it widely expressed. Meltrin‐α mRNA was readily detected in the osteoblast, the most abundant cell type in bone. In situ hybridization analysis on sections of neonatal mice revealed high levels of expression in the trabecular meshwork of long bones, the basal regions of the dermis and its underlying mesenchyme. We conclude that expression of meltrin‐α mRNA is not restricted to fusagenic cells and that, in bone, the osteoblast is the major source. J. Cell. Biochem. 67:136–142, 1997.