Obesity is a grave disease that is increasing in global prevalence. Aberrant neuronal DNA methylation patterns have been implicated in the promotion of obesity development, but the role of neuronal DNA methyltransferases (Dnmts; enzymes that catalyze DNA methylation) in energy balance remains poorly understood. We investigated the role of neuronal Dnmt1 in normal energy regulation and obesity development using a novel Dnmt1 knockout mouse model, Dnmt1fl/fl Synapsin1Cre (ND1KO), which specifically deletes Dnmt1 in neurons. ND1KO and fl/fl control littermates were fedeither a standard chow diet or a high fat diet (HFD). We conducted a deep analysis to characterize both peripheral and central aspects of the ND1KO phenotype. We found that neuronal Dnmt1 deficiency reduced adiposity in chow-fed mice and attenuated obesity in HFD-fed male mice. ND1KO male mice had reduced food intake and increased energy expenditure on the HFD. Furthermore, these mice had improved insulin sensitivity as measured by an insulin tolerance test. HFD-fed ND1KO mice had smaller fat pads and an upregulation of thermogenic genes in brown adipose tissue. These data suggest that neuronal Dnmt1 deletion increased diet-induced thermogenesis, which may explain the lean phenotype in HFD-fed ND1KO mice. Interestingly, we found that ND1KO male mice had elevated estrogen receptor-α gene expression in the hypothalamus, which previously has been shown to control body weight. Immunohistochemistry experiments revealed that estrogen receptor-α (ERα) protein expression was upregulated in the dorsomedial region of the VMH (VMHdm), a region which may mediate the central effect of leptin. Finally, we tested whether ND1KO mice had reduced methylation of the ERα gene promoter, which might explain the ERα upregulation. Neuronal Dnmt1 deficiency decreased methylation at two CpG sites on Exon A in chow-fed mice. Collectively, these data suggest that neuronal Dnmt1 regulates energy homeostasis through pathways controlling food intake and energy expenditure, and that ERα in the VMHdm may mediate these effects.

Breast cancer is the second leading cause of cancer-related death in women. Mutations in the tumor suppressor gene BRCA-1 confer a high risk of breast tumor development. However, in sporadic breast cancers, which represent 90-95% of breast cancer cases, BRCA-1 expression is downregulated in the absence of mutations in the BRCA-1 gene. This suggests that epigenetic effectors may contribute to disruption of BRCA-1 expression and the onset of mammary tumors.Prototypical environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to alter mammary gland development, act as endocrine disruptors and tumor promoters. Population studies detected accumulation of TCDD in women's adipose tissue and breast milk. Moreover, sporadic breast tissue exhibited statistically significant higher levels of PAH-DNA adducts. Based on this information, we examined the effect of B[a]P on the tumor suppressor BRCA-1and observed that exposure to B[a]P led to repression of BRCA-1 transcription through a p53-dependent mechanism. We have also demonstrated that 17β-estradiol (E2) stimulated the recruitment of ERα and AP-1 family members to a region of the BRCA-1 promoter flanking an AP-1-like site. However, accumulation of p53 prevented E2-mediated BRCA-1 transcription and recruitment of ERα, potentially providing one mechanism of B[a]P-mediated repression.In addition, the effects of B[a]P and TCDD are mediated through binding of the liganded aromatic hydrocarbon receptor (AhR) to dioxin or xenobiotic-responsive elements (XRE). We have evidence that suggests B[a]P and TCDD may modulate repression of E2-stimulated BRCA-1 expression through 1) binding of the liganded AhR to XREs on the BRCA-1 promoter and 2) preventing promoter occupancy by p300 and SRC-1.Taken together, the data presented here suggest that the transcriptional regulation of BRCA-1 is complex and involves modulation of the recruitment of ERα, AhR, p53, and their cofactors. An important implication of these findings is a greater understanding of the role of ERα, AhR, and p53 in regulation of BRCA-1 which could lead to the development of therapeutic strategies that target these interactions to enhance upregulation of BRCA-1 expression in sporadic breast tumors.

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.

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

No description available.

Approximately 70% of breast cancers are estrogen receptor positive, of which 40% have ER mutations. Some of these mutations occur in the three intrinsically disordered regions (IDRs) of estrogen receptor-α (ERα). It is not fully understood how these mutations affect the function of ERα, however they have been suggested to affect DNA and ligand binding, transcriptional activation and dimerization. In this study, we used mammalian one-hybrid (M1H) assays to determine the effect of deletions and mutations in the IDRs on the activation of ERα. We found that deletions in the N- and C-terminal IDRs and 7/11 patient mutations tested decreased the transcriptional activation of ERα in the presence of 17β-estradiol (E2). Interestingly, a 23x glycine-serine replacement of the hinge IDR replacement resulted in a 7-fold increase in activation. Our results show that IDRs are important for full transcriptional activation and regulation of ERα. / 2025-06-23T00:00:00Z

No description available.

It is well established that the 17-estradiol is involved in the development and homeostasis of reproductive and extra-reproductive tissues, particularly the mammary gland. Estradiol classically binds to Estrogen Receptor (ERα), which is a member of the nuclear receptor superfamily. ER mediates nuclear (transcription) and plasma membrane (signaling) ERα function. Interestingly, the membrane initiated steroid signaling (MISS) required a post translational modification of the receptor: palmitoylation of the human Cys-447 or the murine Cys-451 counterpart. The main objectives of my PhD thesis were to decipher the physiological role of membrane ERα in mammary gland development and to understand how the membrane ER signaling impact breast cancer. To do so, we used the transgenic mouse model C451A-ER in which the single point mutation (C451A) was introduced to abolish palmitoylation of ER (membrane addressing signal). We demonstrate that the point mutation of the palmitoylation site of ER alters the paracrine signaling of luminal epithelial cells and by consequence the repopulation properties of the mammary stem cells. We also studied the involvement of the membrane effects of the Estrogen Receptor ERα in the 17β-estradiol response dose of the mammary gland. Finally, by breeding the C451A-ER mice with the widely used transgenic mice model of tumorigenesis (PyMT), we provide the first evidence that the membrane ERα influences tumorigenesis. These findings pave the way on an unexpected role of non-genomic function of ERα in the mammary gland physiology and physiopathology. / It is well established that the 17-estradiol is involved in the development and homeostasis of reproductive and extra-reproductive tissues, particularly the mammary gland. Estradiol classically binds to Estrogen Receptor (ERα), which is a member of the nuclear receptor superfamily. ER mediates nuclear (transcription) and plasma membrane (signaling) ERα function. Interestingly, the membrane initiated steroid signaling (MISS) required a post translational modification of the receptor: palmitoylation of the human Cys-447 or the murine Cys-451 counterpart. The main objectives of my PhD thesis were to decipher the physiological role of membrane ERα in mammary gland development and to understand how the membrane ER signaling impact breast cancer. To do so, we used the transgenic mouse model C451A-ER in which the single point mutation (C451A) was introduced to abolish palmitoylation of ER (membrane addressing signal). We demonstrate that the point mutation of the palmitoylation site of ER alters the paracrine signaling of luminal epithelial cells and by consequence the repopulation properties of the mammary stem cells. We also studied the involvement of the membrane effects of the Estrogen Receptor ERα in the 17β-estradiol response dose of the mammary gland. Finally, by breeding the C451A-ER mice with the widely used transgenic mice model of tumorigenesis (PyMT), we provide the first evidence that the membrane ERα influences tumorigenesis. These findings pave the way on an unexpected role of non-genomic function of ERα in the mammary gland physiology and physiopathology.

Thesis (Ph. D.)--University of Virginia, 2008. / [a] in title is the Greek letter alpha. Includes bibliographical references. Also available online through Digital Dissertations.

Background and aims of the study. Prostate cancer (PCa) is one of the most common hormone-dependent cancers in men in Western and also Asian countries. The standard treatment options for localized PCa include surgery and androgen-deprivation therapy (ADT). However, most patients upon ADT therapy invariably relapse and progress to a more aggressive and metastatic stage termed as castration-resistant PCa (CRPC). Accumulating studies indicate that androgen receptor (AR) transcriptional activity is dysregulated during the advanced progression of CRPC. One important mechanism responsible for the growth of CRPC includes increased intra-tumoral androgen synthesis in PCa. Recently, a novel androgen-responsive fusion gene TMPRSS2:ERG formed by fusion between the transmembrane protein TMPRSS2 and transcription factor ERG, has been identified in approximately 50% PCa samples, which results in the aberrant expression of ERG function as oncogenic factor in PCa. Currently, TMPRSS2:ERG is regarded as a significant potential diagnostic and prognostic biomarker for PCa. Estrogen-related receptor alpha-ERRα, the first identified ligand-independent orphan nuclear receptor, is characterized to be up-regulated in advanced cancers, suggesting that ERRα might play important regulatory roles in the malignant progression of PCa. Previous studies showed that ERRα can functionally cross-talk with AR signaling via co-targeting to AR targets and regulate the expression of some steroidogenic enzymes in breast cancer. Based on this background, it is hypothesized that ERRα could functionally regulate the TMPRSS2:ERG fusion gene and play a regulatory role in the development and progression of CRPC through activation of the intracellular androgen synthesis pathway. / Results. 1) The results obtained in this study showed that suppression of ERRα by its specific inverse agonist XCT790 or shRNA-knockdown could induce down-regulation of TMPRSS2:ERG and also its target genes in AR-positive VCaP PCa cells. 2) Ectopic expression of ERRα and/or its coactivator PGC-1α could increase the expression of TMPRSS2:ERG in AR-negative NCI-H660 PCa cells. 3) Two ERRα-DNA binding elements were identified by ChIP assay and sequence analysis in the promoter of TMPRSS2:ERG and both of these two elements could be transactivated by ERRα and PGC-1α. 4) Ectopic expression of TMPRSS2:ERG under the regulation of ERRα enhanced the prostatic cell invasion capacity as shown in the TMPRSS2:ERG infectants of BPH-1 and PC-3 prostatic cells. 5) ERG expressed by the TMPRSS2:ERG fusion could directly transactivate the ERRα gene in prostatic cells. 6) A positive correlation on the expressions between TMPRSS2:ERG and ERRα was demonstrated in a xenograft model of CRPC (VCaP-CRPC). 7) The expression of TMPRSS2:ERG and ERRα showed significant up-regulation and the transactivation activity of ERRα was also enhanced in castration-resistant VCaP-CRPC cells. 8) Ectopic expression of ERRα could promote resistant growth capacity to androgen-deprivation condition in LNCaP PCa cells, whereas shRNA-mediated silence of ERRα could weaken this resistant capacity. Furthermore, ectopic expression of ERRα in LNCaP-ERRα infectants could promote their in vivo growth resistance to castration in SCID mice. 9) Expression of several androgenic enzyme genes, including CYP11A1, CYP17A1 and ARK1C3, were detected to be up-regulated in castration-resistant VCaP-CRPC cells. Moreover, ectopic expression of ERRα could induce the increased expression of these enzyme genes in LNCaP-ERRα infectants, whereas knockdown of ERRα by shRNA could decrease their expression. 10) ERRα could directly transactivate the gene promoters of CYP11A1, CYP17A1 and ARK1C3 which contain ERRE elements prediction by sequence analysis. These results suggested that ERRα could play a role in de novo or intra prostatic androgen synthesis in the PCa cells. / Conclusions. The results obtained in this study suggested that ERRα and TMPRSS2:ERG could form a positive reciprocal loop in PCa cells, and ERRα could also promote the resistant growth capacity of PCa cells resistant to the androgen-deprivation condition in vitro and also castration-resistant growth in vivo via a mechanism of up-regulation of androgenic enzyme genes. The results also suggested that ERRα might play a significant regulatory role in the development and progression of PCa, particularly the advanced CRPC, and also ERRα could be a potential therapeutic target for the treatment of PCa, particularly the advanced PCa-CRPC. / 研究背景與研究目的：前列腺癌作為激素依賴的一種癌症，經常出現在西方和亞洲國家的男性人群中。對於局限性前列腺癌多採用外科手術和去勢的治療。但是大多數病人經過去勢治療后會再次復發並且形成更加惡心幾轉移的前列腺癌，稱之為去勢難治性前列腺癌（CRPC）。越來越多的研究表明在去勢難治性前列腺癌發病過程中，雄激素受體轉錄活性異性增強。其中一個重要機理解釋為前列腺癌細胞自身合成的雄激素增多。進來，在大約50%的前列腺癌病人中新檢測到一個受雄激素受（AR）體調控的融合基因TMPRSS2:ERG，它是由稱為TMPRSS2的一個跨膜蛋白和一個稱為ERG的轉錄因子融合而成，它的出現導致了在前列腺癌中異常的稱為致癌因子的ERG蛋白的高表達。目前，TMPRSS2:ERG已經被作為一個重要的潛在的診斷和預測的標誌物應用在前列腺癌中。作為第一個鑒定的配體不依賴的孤兒受體-ERRα，被證明在晚期的癌症中有很高的表達，預示著ERRα可能在惡性的癌症中起到一個非常重要的調控作用。之前的研究表明通過共同調控AR的下游基因，ERRα同AR信號通路之間有功能性的交叉調控；除此之外，在乳腺癌中，ERRα還可以調控一些類固醇類化合物的合成相關的一些酶的合成。依據上述，我們推定ERRα可能功能性地調控TMPRSS2:ERG融合基因的表達並且通過調控細胞內的雄激素的合成進而在去勢難治性前列腺癌的發生和發展中起到一個非常重要的作用。 / 結果：本論文研究結果總結如下：1）在有AR表達的前列腺癌細胞-VCaP細胞中，通過ERRα特異性的抑制劑XCT790處理或者shRNA介入的干擾ERRα的mRNA的方法來抑制ERRα，下調了TMPRSS2:ERG和它的一些下游調控基因的表達。2）在沒有AR表達的前列腺癌細胞-NCI-H660細胞中，上調ERRα或者它的特異性的共激活因子PGC-1α表達可以提升TMPRSS2:ERG的表達。3）通過ChIP實驗，在TMPRSS2:ERG的啟動子上面，兩個ERRα的DNA結合位點被鑒定出來。並且這兩個位點可以被ERRα和PGC-1α轉錄激活。4）在兩個前列腺細胞BPH-1和PC-3細胞中，在ERRα的調控下高表達TMPRSS2:ERG融合基因可以增強細胞的侵襲能力。5）融合基因TMPRSS2:ERG導致的ERG蛋白的表達可以直接轉錄激活ERRα的表達。6）我們通過VCaP細胞的異種移植建立VCaP-CRPC的體內模型來模擬CRPC過程，在整個過程中，我們發現TMPRSS2:ERG和ERRα有一致性的表達相關性。除此之外，我們根據上述動物模型通建立了VCaP-CRPC細胞系，並且發現在VCaP-CRPC細胞細胞中，TMPRSS2:ERG和ERRα都有被上調並且ERRα的轉錄活性同樣也提升。7）在LNCaP細胞中高表達ERRα可以提升細胞在去除雄激素的環境中生長的能力。但是當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減弱這種生長的能力。用LNCaP-ERRα穩轉ERRα的細胞異種移植建立SCID老鼠體內腫瘤模型，我們發現和LNCaP-pBABE對照組相比，LNCaP-ERRα細胞生長的更快更大。並且在對老鼠進行睪丸切除術后，LNCaP-ERRα組細胞更快適應這種環境并繼續生長，相比之下，LNCaP-pBABE對照組則持續萎縮減小。8）在上述的VCaP-CRPC細胞中，我們發現一些和雄激素合成相關的關鍵的酶包括CYP11A1，CYP17A1和ARK1C3的表達量有顯著地提升。而且在LNCaP-ERRα細胞中同樣檢測到這些酶的表達量的提升。然而當在LNCaP細胞中用shRNA干擾掉ERRα可以明顯減降低上述酶的表達。9）我們在CYP11A1，CYP17A1和ARK1C3基因的啟動子區域發現有ERRα結合位點，並且發現這些位點可以被ERRα轉錄激活。 / 結論：本論文的研究結果提示在前列腺癌細胞中，ERRα和TMPRSS2:ERG可以形成一個相互正向調控的循環。除此之外，上調ERRα可以促進細胞在去除雄激素的環境中生長的能力，並且在動物體內可以提升細胞在睪丸去除的環境中的適應和生長能力。這種體內和體外的能力的提升是通過一種潛在的上調前列腺癌細胞的雄激素合成相關的關鍵的酶的表達，進而提升雄激素的含量而得以實現的。上述的結果預示著ERRα可能在前列腺癌發生機發展的過程中起到非常重要的調控作用，尤其在晚期的CRPC中。同時，ERRα也可能作為一個潛在的重要的前列腺癌尤其是晚期的CRPC的治療靶點，尤其是一些潛在ERRα的特異性抑制劑，比如XCT790，可能作為將來用以作為治療前列腺癌的特異性靶點藥物。 / Xu, Zhenyu. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 126-143). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Xu, Zhenyu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.