Faculty of Science School of Anatomical Sciences 9901061h karrenthomson@yahoo.co.uk / Clomiphene citrate (CC), a synthetic estrogen, is an efficient superovulator used in infertility treatment. However pregnancy rates resulting from CC treatment are low. Research has suggested that this may be due to an aberrant effect on implantation; CC binds to estrogen receptors (ER) and may affect estrogen responsive gene expression and thus implantation. This study investigates the effect of CC on ERa, 90kDa heat shock protein (Hsp90) and Hoxa10 expression in the rat uterus. Hsp90 binds to ERa in the absence of ligand and is involved in inducing a high affinity ligand binding conformation in the ER and in transactivation of the ER. Hoxa10 has been shown to be essential for uterine receptivity to implantation. CC (0.25mg) was given to ovariectomized rats, either alone or prior to a hormonal regime known to induce uterine receptivity for implantation. Expression of ERa, Hsp90 and Hoxa10 was determined by Western blotting, fluorescence immunocytochemistry and reverse transcription polymerase chain reaction. The single dose CC treated rats were compared to the controls as well as to ovariectomized rats treated with 0.5mg 17b estradiol (E2). The CC treated pseudopregnant rats (CCPPPE treated) were compared to 5½ day pregnant and pseudopregnant rats without CC (PPPE treated), to determine CCs effect at implantation. E2 upregulated ERa and Hsp90 expression in the rat uterus compared to controls (p<0.05). The finding for ERa was unexpected as other studies have shown that E2 decreases ERa levels a few hours after administration in the uterus. The present study therefore suggests a biphasic effect of E2 on ERa expression in the rat uterus. The effect of E2 on Hsp90 and ERa also proposes a balance between the levels of these two proteins in the uterus, to keep ERa in its optimal state and suggests that too high and too low a concentration of Hsp90 may both be inhibitory to ERa functioning. No significant difference was found in ERa and Hsp90 expression between the non-receptive (vehicle treated) and the receptive (PPPE treated) rat uteri, suggesting that these two genes are not markers for receptivity. However E2 is known to induce implantation of donor blastocysts in progesterone (P4) primed uteri. Therefore it is still essential for ERa to be present at implantation. It is of interest that CC downregulated ERa levels both in ii the absence of ovarian hormones and at implantation in the rat uterus. It is therefore proposed that this antiestrogenic effect would render the uterus less sensitive to the E2 required to induce implantation, thus accounting for low pregnancy rates with CC use. Although CC did not alter the expression of Hsp90 in this study, the reduction in ERa levels in response to CC may also upset the balance in the expression of these two genes, which may affect the transcriptional activity of ERa, and further prevent implantation. No clear results were obtained for Hoxa10 expression with the Western blots. However based on the ICC results, CC did not appear to affect Hoxa10 expression. Since P4 and not E2 is known to have the predominant effect on Hoxa10 expression, it is likely that E2 analogs, such as CC, would also not affect Hoxa10 expression to a significant degree. Future work will aim to separate the different uterine compartments and to determine the effects of CC on the expression of other implantation specific genes in the uterus.

<p>Estrogens and the estrogen receptor (ER) have been implicated in the etiology of breast cancer and osteoporosis. However, the mechanisms by which this receptor-ligand complex manifest their regulatory activities in these processes is not completely understood. The development and subsequent definition of the molecular mechanism of action of selective ER modulators (SERMs), compounds with differential relative agonist/antagonist activity, has uncovered an unanticipated complexity in this signaling pathway. Furthermore, these analyses indicat that it is likely that in addition to the classical steroidal estrogens, which exhibit agonist properties, endogenous compounds exist that interact with ER and function as physiological SERMs. Recently, 27-hydroxycholesterol (27HC) was identified as an endogenous ER ligand with tissue-specific estrogenic/anti-estrogenic activities. Indeed, we determined that 27HC exhibited the three basic properties of a SERM: 1) it bound competitively with estradiol (E2) to both genetic subtypes of ER, ER&#945; and ER&#946;; 2) it induced a unique conformation of ER that is likely related to its biological activity; and 3) it displayed tissue-specific ER modulatory activity in the cardiovascular system, breast, and bone. In particular, we undertook a series of in vivo studies to show that a pathological elevation of 27HC was associated with decreased bone quantity, an effect that was partially rescued by E2 supplementation. The ability of 27HC to decrease bone density in the absence of endogenous estrogens suggests that the circulating level of 27HC may be of critical importance in determining osteoporosis risk in post-menopausal women. Interestingly, cholesterol-lowering statins have been shown to improve bone density; thus, given the stoichiometric relationship between circulating cholesterol and 27HC, our data provide a possible explanation for the observed bone sparing actions of this class of drugs. </p><p>In general, it is considered that SERM activity can be explained by the ability to induce differential alterations in ER structure and the impact that this has on the recruitment of functionally distinct cofactors. The results of our studies reveal a much more complex picture and suggest that some SERM pharmacology can be ascribed to actions in pathways that do not include ER. Specifically, we have determined that the SERM 4-hydroxy-tamoxifen (4OHT) can bind to and activate the aryl hydrocarbon receptor (AHR). Given that AHR controls the expression of E2-metabolizing enzymes, our finding that 4OHT regulates AHR in the context of breast cancer could have important pharmacological and pathological implications. Interestingly, our preliminary in vitro data indicate that the ability of 4OHT to inhibit osteoclast (OC) differentiation, and thus aid in preserving bone density in post-menopausal women, is primarily dependent on expression of AHR, not ER. Conversely, the inhibitory activity of raloxifene (RAL), another SERM, on OC differentiation was absolutely dependent on ER. Thus, the activity of 4OHT in bone is likely to be a composite response requiring its actions on both ER and AHR. </p><p>Many new aspects of the estrogen and ER signaling pathways have been uncovered as we learn more about ligands that modulate ER by altering its conformation and thus its ability to engage in protein-protein interactions. Collectively, our findings demonstrate that the intersection between cholesterol metabolism, ER signaling, and the AHR pathway will have important consequences in regulating cellular function, and may be involved in the development or progression of multiple disease states.</p> / Dissertation

This dissertation focused on two classes of molecules implicated in processes of implantation and placentation in sheep and pigs. Study one examined the temporal/spatial distribution of several Small Integrin-Binding Ligand, N-Linked Glycoprotein (SIBLING) family members in cyclic and pregnant ovine uterus. Studies two and three evaluated the relationships between progesterone (P4) and estrogen (E2) and their receptors (PGR and ESR1, respectively) on FGF7 mRNA expression within the endometrium and placenta of pigs. Study one showed that dentin sialophosphoprotein (DSPP) was first detected in luminal epithelium (LE) of Day 15 cyclic and pregnant sheep. Stromal expression of DSPP was first detected on Day 20 of pregnancy in stratum compactum and remained prominent in stroma through Day 120. Stromal DSPP protein was positively influenced by the conceptus based upon analysis of a unilaterally pregnant ewe model system. Immunoreactive dentin matrix protein 1 (DMP1), matrix extracellular phospoglycoprotein (MEPE) were localized to the stroma of cyclic and pregnant sheep, however, these proteins appeared to be constitutively expressed. BSP was not detected in ovine endometrium. Study two determined the effects of E2, P4, P4+E2, P4+the PGR antagonist (ZK137, 316), and P4+E2+ZK on FGF7 mRNA expression in uterine LE of ovariectomized pigs. Results indicate that P4 is permissive to FGF7 mRNA expression by down-regulating PGR in LE; P4 stimulates PGR-positive uterine stromal cells to release an as yet unidentified progestamedin that induces FGF7 mRNA expression by LE; E2 and P4 can induce FGF7 mRNA in the absence of PGR rendered nonfunctional by ZK. Study three showed the expression of ESR1, PGR and FGF7 in the uterine and placental tissue of pregnant pigs from Day 20 through 85. Results reveal a positive correlation between stromal cell expression of PGR and FGF7 mRNA which suggests that P4 is permissive to FGF7 mRNA expression by down-regulating PGR in LE. FGF7 mRNA in later pregnancy is maintained by the release of progestamedin from PGR-positive stromal cells. A novel finding was the presence of ESR1 in porcine placenta on Days 20 through Day 85 of pregnancy suggesting that E2 may play important roles in the placental biology of the pig.

Breast cancer is the most common malignancy affecting women and the second leading cause of death among women in the United States. Alcohol consumption is one of the few modifiable risk factors for breast cancer development but the mechanism by which it contributes to mammary cancer development and progression remains unclear, although it has been suggested that estrogen is critical for this process. To determine if alcohol promotes mammary tumor development via the estrogen pathway, estrogen receptor alpha-negative (ER[alpha]-negative) MMTV-neu mice were treated with various doses of ethanol and activation of estrogen signaling was measured. Our results showed that alcohol consumption increased estrogen signaling activation, serum estrogen levels and, most interestingly, expression of ER[alpha] in tumor tissue in the ER[alpha]-negative mice. Several lines of evidence in literature suggest that ER[alpha] expression in ER[alpha]-negative cancer cells is inhibited through epigenetic regulation. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than DNA sequence changes. Thus, to determine whether alcohol may regulate ER[alpha] re-expression in ER[alpha]-negative breast cancer cells through epigenetic mechanisms, we examined the effects of ethanol on CpG methylation and histone modifications (acetylation and methylation) of two ER[alpha]-negative breast cancer cell lines, MDA-MB-231 (human) and MMTV-neu (mouse). We also examined whether the epigenetic modifications subsequently affect the recruitment of transcriptional regulation complexes to the ER[alpha] promoter to regulate ER[alpha] transcription. Results showed that alcohol promotes ER[alpha] re-expression in these ER[alpha]-negative cell lines and that this effect was associated with decreased CpG methylation, an overall increase of histone acetylation and decrease of histone methylation, and an alteration in the enrichment of the ER[alpha] transcriptional regulation complexes (pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1) at the ER[alpha] promoter, which may contribute to cancer cell progression. In addition, we found that the inhibition of ER[alpha] by tamoxifen specifically blocks the effects of alcohol on ER[alpha] reactivation. To determine how alcohol promotes cell invasive ability, a critical process for cancer progression, we examined the role of two genes, metastasis suppressor Nm23 and integrin alpha-5 ITGA5, which we identified to be important for alcohol-induced breast cancer cell invasion. It has previously been shown that estrogen may regulate Nm23 expression and that estrogen regulation may be important for ITGA5-mediated cancer progression. Our results showed that alcohol promotes cancer cell invasion through the down-regulation of Nm23, which led to the subsequent increase of ITGA5 and increase of cell invasion. Collectively, data from my research strongly supports and provides evidence that alcohol promotes breast cancer development and progression through the regulation of estrogen signaling. / text

The steroid receptor RNA activator gene (SRA1) generates two distinct entities. SRA RNA coactivates several NRs whereas SRA protein (SRAP) is suspected to regulate the activity of several transcription factors, including estrogen receptors (ER). Splicing of SRA intron-1 is the major event defining SRAP coding frame. Fully spliced, coding SRA and intron-1 retained, non-coding SRA coexist in breast cancer cells. The relative proportion between the two types of SRA RNA maintains a balance between two genetically linked entities, SRA and SRAP. In this study, a minigene model was used to demonstrate that the primary sequence of SRA exon-1-intron-1-exon-2 is sufficient for alternative splicing of SRA intron-1. In addition, a modified oligoribonucleotidic construct promotes SRA intron-1 retention in breast cancer cells. This oligoribonucleotide differentially alters estradiol-induced transcription of ER regulated genes. Together, results presented herein demonstrate that the SRA-SRAP balance, which can be artificially modified by targeting alternative splicing of SRA intron-1, might be a new critical target to treat breast cancer patients.

The steroid receptor RNA activator gene (SRA1) generates two distinct entities. SRA RNA coactivates several NRs whereas SRA protein (SRAP) is suspected to regulate the activity of several transcription factors, including estrogen receptors (ER). Splicing of SRA intron-1 is the major event defining SRAP coding frame. Fully spliced, coding SRA and intron-1 retained, non-coding SRA coexist in breast cancer cells. The relative proportion between the two types of SRA RNA maintains a balance between two genetically linked entities, SRA and SRAP. In this study, a minigene model was used to demonstrate that the primary sequence of SRA exon-1-intron-1-exon-2 is sufficient for alternative splicing of SRA intron-1. In addition, a modified oligoribonucleotidic construct promotes SRA intron-1 retention in breast cancer cells. This oligoribonucleotide differentially alters estradiol-induced transcription of ER regulated genes. Together, results presented herein demonstrate that the SRA-SRAP balance, which can be artificially modified by targeting alternative splicing of SRA intron-1, might be a new critical target to treat breast cancer patients.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 87-95).

Estrogen is an important regulator and promoter of epithelial wound healing. This is facilitated by increased keratinocyte and fibroblast migration and proliferation, as well as promotion of angiogenesis, matrix deposition and inflammatory response dampening. The potential to target this pathway for therapeutics is highlighted by observations that post-menopausal women on hormone replacement therapy have a significantly lower incidence of venous ulcers. Previous work from this laboratory identified four SNPs (single nucleotide polymorphisms) in the 5’UTR of estrogen receptor beta (ERβ) gene that are associated with venous ulcer predisposition. Disease association is further supported by the identification of ERβ as the main conduit of the beneficial effects of estrogen signalling on wound healing. SNP’s of the 5’UTR can affect transcriptional expression through the modification of transcription factor binding sites, epigenetic modifications and translational efficiency via mRNA localisation and secondary structure alterations. To investigate the possible biological function of these SNPs, we have developed disease relevant cell based assays where primary keratinocyte and fibroblast cells were selected harbouring disease-associated SNPs. We demonstrate that the presence of venous ulcer-associated ERβ SNPs reduced the expression of ERβ in skin cells and reduced their migration and proliferative capabilities. Evidence gathered here suggests that ERβ expression is curtailed by a change in transcription factor binding, likely facilitated by the change in nucleotide sequence brought about by the rs2987983 SNP. Further, we demonstrate that SNP-induced changes in fibroblast expression of growth factors and inflammatory mediators can hinder keratinocyte migration and induce a pro-inflammatory phenotype in human monocytes. Lastly, RNAseq analysis of keratinocytes reveals a SNP-dependant gene expression profile that is detrimental to wound healing. This work provides the first evidence of a direct functional link between venous ulcer-associated ERβ SNPs and dysfunctional wound healing. Investigating ERβ SNPs has provided insight into novel mechanisms of estrogen signalling that can be applied for therapeutic development to treat venous ulcers.

Accumulating evidence from epidemiological, wildlife, and laboratory studies indicates that abnormalities of reproduction, development and physiology can be ascribed to environmental contaminants with biological activities. Many such contaminants disrupt essential hormone-regulated processes by virtue of their ability to interact with nuclear hormone receptors (endocrine disrupting chemicals, EDC). Of particular concern are chemicals that mimic or block estrogen signaling (xenoestrogens, XE) through their direct interaction with estrogen receptors (ER). The current model of XE action focuses on disrupted transcriptional activity, as measured by changes in the expression of ER-regulated genes. However, transcription is tightly coupled to splicing, by which a single target gene transcript is processed to multiple structurally and functionally different mRNAs. In theory, any XE that interacts with ER to regulate transcription has the potential to disrupt splicing, thereby affecting not only mRNA quantity but also quality. To address this hypothesis, alternative splicing of the gene encoding ER alpha (esr1), itself an estrogen responsive gene, was investigated. In these studies, killifish (Fundulus heteroclitus), an environmentally relevant species, and zebrafish (Danio rerio), an advantageous laboratory fish model, were used. First, the occurrence of ER alpha splice variants in adult tissues, in developing embryos and in response to estrogens in the two species was documented. Additionally, the effects of long-term, multigenerational XE exposure on ER alpha splicing were examined in two killifish populations, one from an estrogenic (polluted) site and a second population from a reference (unpolluted) site. A subset of ER alpha variants from killifish was expressed in cell culture to document their transcriptional activities. To determine the in vivo relationship between estrogen responsiveness and an ER alpha splice variant of interest, esr1 splicing was experimentally altered in living embryos by microinjecting morpholino oligonucleotides, and changes in induction of a panel of estrogen responsive gene targets were measured as markers of effect. These results provide evidence that dysregulation of mRNA processing is also a mechanism of XE action, and suggest that resultant ER alpha splice variants mediate the short-term effects of estrogen disruption and are also part of the adaptive response to long-term, multigenerational XE exposures in the natural environment.

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No. of bitstreams: 1 Tese - Gecildo Soriano dos Anjos.pdf: 1274691 bytes, checksum: 928de7ba767ef6a088de505cb09254ce (MD5) Previous issue date: 2012-12-11 / Não informada / The goiter is a clinical condition characterized by the increase of the thyroid gland at the expense of a nodule(s), tangible or not, whose incidence in general ranges from 4% to 7%, especially in women. It is endemic in Amazon Region, and a public health problem. Objective: To evaluate the expression of the estrogen receptor (RE) in a qualitative way in surgical pieces with the histopathologic diagnosis of goiter patients undergoing thyroidectomy in Manaus city. Methods: Forty-nine patients with goiter histopathological diagnosis were selectec, and from its paraffin blocks, was evaluated the expression of the estrogen receptor (not specific) by immunohistochemistry using the technique of streptoavidina-biotin immunoperoxidase staining and primary antibodies anti-ER. The statistical analysis used the non-parametric test Fisher's exact, with 5% significance level, and Pearson's correlation coefficient. Results: Forty-seven (95.9%) patients were negative for estrogen receptor, while two (4.1%) had positive immunoexpression. Thirty eight (84.45%) patients were female and seven (15.6%) were male. The majority of patients were in the age range 42 to 52 years (19 patients), while the minority (two patients) were above 74 years. The minimum patients age was 31 years and the maximum was 87 years, with an average of approximately 53 years. No statistical difference was found (p-value: 0.732) to correlate the gender of the patients with positive immunoexpression of estrogen receptor. There was a reasonable correlation negative (Pearson Correlation - 13.9%) between immunohistochemical analysis and the patients age, in which the two patients ER positive were within the range age (42 to 52 years) of higher incidence (42.6% of patients). Conclusion: there is not a direct relationship between the goiter development and the presence of the estrogen receptor in the thyroid gland. / O bócio é uma condição clínica caracterizada pelo aumento da glândula tireoide à custa de nódulo(s), palpável ou não, cuja incidência, em geral varia de 4% a 7%, principalmente em mulheres. É endêmico na Amazônia, e problema de saúde pública. Objetivo: Avaliar a imunoexpressão do receptor de estrogênio (RE) de forma qualitativa em peças cirúrgicas com o diagnóstico histopatológico de bócio de pacientes submetidos à tireoidectomias na cidade de Manaus/AM. Métodos: Selecionaram-se quarenta e nove pacientes com diagnóstico histopatológico de bócio e, a partir de seus blocos de parafina, foi avaliada a expressão do receptor de estrogênio (não específico) por imuno-histoquímica com a técnica da streptoavidinabiotina- imunoperoxidase e anticorpos primários anti-RE. A análise estatística utilizou o teste não paramétrico exato de Fisher, com nível de significância de 5%, e o Coeficiente de correlação de Pearson. Resultados: Quarenta e sete (95,9%) pacientes mostraram-se negativos para o receptor de estrogênio, enquanto que dois (4,1%) apresentaram imunoexpressão positiva. Trinta e oito (84,45%) pacientes eram do sexo feminino e sete (15,6%) do gênero masculino. A maioria dos pacientes encontravam-se na faixa etária de 42 a 52 anos (19 pacientes), enquanto que a minoria (dois pacientes) estavam acima de 74 anos de idade. A idade mínima entre os pacientes foi de 31 anos e a idade máxima de 87 anos, com média de aproximadamente 53 anos. Não foi encontrada significância estatística (p-valor: 0,732) ao correlacionar-se o gênero dos pacientes com a imunoexpressão positiva do receptor de estrogênio. Houve uma correlação razoável negativa (Correlação de Pearson – 13,9%) entre a análise imuno-histoquímica e a idade dos pacientes, no qual os dois pacientes RE positivos encontravam-se dentro da faixa etária (de 42 a ix 52 anos) de maior incidência (42,6% dos pacientes). Conclusão: não existe uma relação direta entre o desenvolvimento do bócio e a presença do receptor de estrogênio na glândula tireoide; não foi possível correlacionar a expressão do receptor de estrogênio com o gênero dos pacientes devido à baixa casuística dos pacientes masculinos (sete) comparados com os femininos (42).