Estrogens and selective estrogen receptor modulators regulate gene and protein expression in the mesenteric arteries.
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ABSTRACT:
SUBMITTER: Mark-Kappeler CJ
PROVIDER: S-EPMC3159742 | biostudies-literature | 2011 Jul-Sep
REPOSITORIES: biostudies-literature
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Estrogens and selective estrogen receptor modulators regulate gene and protein expression in the mesenteric arteries.
Mark-Kappeler Connie J CJ Martin Douglas S DS Eyster Kathleen M KM
Vascular pharmacology 20110601 1-3
Estrogen has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Gene targets of estrogen and SERMs in the vasculature are not well-known. Thus, the present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculatur ...[more]
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Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation. Goal of the experiment: To examine the effects of three formulations of estrogen (ethinyl estradiol, equilin, and estradiol benzoate) and of the selective estrogen receptor modulators, tamoxifen and raloxifene, on gene expression in the mesenteric arteries of the rat. Keywords: rat, artery, gene expression, mesentery, estrogen, tamoxifen, raloxifene Experimental factors: hormone treatment, drug treatment Experimental design: Prepubertal female Sprague Dawley rats (Harlan, Indianapolis, IN) were ovariectomized at 4 weeks of age, prior to the initiation of regular ovarian cycles. The animals were allowed to recover for 2 weeks before the experimental protocol was initiated. The rats were randomly divided into 6 groups (n=3-4/group) and treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After 3 weeks of treatment, the rats were euthanized and the mesentery obtained. Total RNA was extracted from each individual sample and used for the synthesis of labeled cRNA for microarray analysis. After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated and harvested and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Mesenteric artery RNA was extracted and purified using an RNeasy Mini Kit (Qiagen). Any potentially contaminating DNA was removed by treatment with RNase-free DNase (Qiagen) on the RNeasy column. Total RNA was quantified and RNA integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA). Following analysis, the RNA was stored at -80ºC until it was used for gene expression studies. DNA microarray: CodeLink Whole Rat Genome Bioarrays (Applied Microarrays, Tempe, AZ) were used. Five μg total RNA was used from each mesenteric arterial sample for the microarray analysis. Complementary RNA was synthesized per company protocol (Ambion). A set of bacterial RNAs was added to each total RNA sample and carried through the synthesis and labeling reactions as controls and for nor¬malization of the data. The microarrays were scanned with the GenePix 4000B (Axon Instruments, Union City, CA) with GenePix Pro 5.0 software (Axon Instruments). Analysis of the microar¬rays was performed with the CodeLink Expression Analysis software (Applied Microarrays) and Acuity 3.1 software (Axon), and statistical analysis was performed with GeneSpring 7.1 software (Agilent, Santa Clara, CA). A sample of n=3 was completed with the microarrays for each treatment. Quality control steps: The cRNA that was synthesized from each mesenteric artery treatment sample was used for hybridization to a single CodeLink (Applied Microarrays) whole rat genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were used. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were isolated mesenteric arteries from individual Sprague Dawley rats that had been treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). Manipulations of biological samples and protocols used: Ovariectomized female rats were treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated, harvested, and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Experimental factor: hormone treatment, drug treatment Technical protocols: The mesenteric arteries were minced with scalpels and TRI reagent (MRC) was added. The tissue was homogenized with a Polytron homogenizer. Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labeled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 5.0 microgram of rat mesenteric artery total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides (CodeLink Whole Rat Genome Bioarrays), and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2011-07-06 | E-GEOD-23037 | biostudies-arrayexpress
Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation.
2011-07-06 | GSE23037 | GEO
Project description:Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1-1?nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ?10?nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1-1?nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.
| S-EPMC6128776 | biostudies-literature
Project description:This study investigates how 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner. We compare the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifen (las) and the phytoestrogen peurarin 15-week old C57BL/6 female mice, either wild type or mice with deletion of ERα-AF1, were ovariectomized and injected subcutaneously one week later with either E2 (115 µg/kg body weight) or olive oil vehicle (Apoteksbolaget, Göteborg, Sweden). Mice were sacrificed after 18 hours of treatment and RNA was isolated from either long bone shafts (flushed femora and tibiae) or vertebral bodies (L5+L6) using an RNAlater reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Gene expression was profiled at the Southern California Genotyping Consortium (SCGC) using MouseRef-8 v2.0 Illumina BeadChips (n=5 mice per group).
2018-04-27 | GSE113732 | GEO
Project description:A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of this gender difference are the sex steroids, estrogen and testosterone. The vasculature is considered to be a site of action of these steroids. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day). The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays were used to identify differential gene expression. Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR. The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone. 3 alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone. Aryl hydrocarbon nuclear translocator-like gene was reduced by testosterone. These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature.
| S-EPMC2080788 | biostudies-literature
Project description:Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) alpha and beta to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERalpha or ERbeta regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERalpha or ERbeta. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERalpha cells synthesized only ERalpha and that U2OS-ERbeta cells expressed exclusively ERbeta. U2OS-ERalpha and U2OS-ERbeta cells were treated either with 17beta-estradiol (E2), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERalpha and U2OS-ERbeta cells by E2, raloxifene, and tamoxifen, respectively. Most genes regulated in ERalpha cells in response to E2, raloxifene, and tamoxifen were distinct from those regulated in ERbeta cells. Only 38 of the 228 (17%) genes were regulated by E2 in both U2OS-ERalpha and U2OS-ERbeta cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERalpha and ERbeta cells. A subset of genes involved in bone-related activities regulated by E2, raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERalpha are distinct from those regulated by ERbeta in response to E2 and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERalpha and ERbeta
| S-EPMC363122 | biostudies-literature
Project description:The estrogen signaling pathway has been reported to modulate prostate cancer (PCa) progression through the activity of estrogen receptors α and β (ERα and ERβ). Given that selective estrogen receptor modulators (SERMs) are used to treat breast cancer, ERs have been proposed as attractive therapeutic targets in PCa. However, many inconsistencies regarding the expression of ERs and the efficacy of SERMs for PCa treatment exist, notably due to the use of ERβ antibodies lacking specificity and treatments with high SERM concentrations leading to off-target effects. To end this confusion, our objective was to study the impact of estrogenic and anti-estrogenic ligands in well-studied in vitro PCa models with appropriate controls, dosages, and ER subtype-specific antibodies. When using physiologically relevant concentrations of nine estrogenic/anti-estrogenic compounds, including five SERMs, we observed no significant modulation of PCa cell proliferation. Using RNA-seq and validated antibodies, we demonstrate that these PCa models do not express ERs. In contrast, RNA-seq from PCa samples from patients have detectable expression of ERα. Overall, our study reveals that commonly used PCa models are inappropriate to study ERs and indicate that usage of alternative models is essential to properly assess the roles of the estrogen signaling pathway in PCa.
| S-EPMC7055213 | biostudies-literature
Project description:Estrogen-based hormone therapy (HT) in postmenopausal women may reduce the risk of Alzheimer's disease (AD), although HT remains controversial. One key concern with HT is the potential of adverse outcomes such as breast and uterine cancer. A promising strategy to maximize HT benefits and minimize HT risks is the use of selective estrogen receptor modulators (SERMs) that exert tissue-specific estrogenic effects. To begin investigating the SERM approach in reducing the risk of AD, we investigated whether AD-like neuropathology in the 3xTg-AD mouse model of AD is regulated by the SERMs propylpyrazole triol (PPT) and diarylpropionitrile (DPN) that exhibit relative specificity for estrogen receptor-alpha and -beta, respectively. Consistent with our previous observations, we found that ovariectomy-induced hormone depletion in adult female 3xTg-AD mice significantly increased accumulation of beta-amyloid protein (Abeta) and decreased hippocampal-dependent behavioral performance. Treatment with 17beta-estradiol (E2) prevented the ovariectomized-induced worsening of both pathologies. PPT treatment was similar to E2 in terms of reducing Abeta accumulation in hippocampus, subiculum, and amygdala but comparatively less effective in frontal cortex. In contrast, DPN did not significantly reduce Abeta accumulation in hippocampus and subiculum, was partially effective in frontal cortex, and nearly as effective as E2 in amygdala. Furthermore, PPT but not DPN mimicked the ability of E2 to improve behavioral performance. These findings provide initial evidence of beneficial actions of SERMs in a mouse model of AD and support continued investigation of SERMs as an alternative to estrogen-based HT in reducing the risk of AD in postmenopausal women.
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Project description:Selective estrogen receptor modulators (SERMs), such as tamoxifen (TAM), have been used extensively for the treatment and prevention of breast cancer and other pathologies associated with aberrant estrogen receptor (ER) signaling. These compounds exhibit cell-selective agonist/antagonist activities as a consequence of their ability to induce different conformational changes in ER, thereby enabling it to recruit functionally distinct transcriptional coregulators. However, the observation that SERMs can also regulate aspects of calcium signaling and apoptosis in an ER-independent manner in some systems suggests that some of the activity of drugs within this class may also arise as a consequence of their ability to interact with targets other than ER. In this study, we demonstrate that 4-hydroxy-TAM (4OHT), an active metabolite of TAM, directly binds to and modulates the transcriptional activity of the aryl hydrocarbon receptor (AHR). Of specific interest was the observation, that in the absence of ER, 4OHT can induce the expression of AHR target genes involved in estradiol metabolism, cellular proliferation, and metastasis in cellular models of breast cancer. The potential role for AHR in SERM pharmacology was further underscored by the ability of 4OHT to suppress osteoclast differentiation in vitro in part through AHR. Cumulatively, these findings provide evidence that it is necessary to reevaluate the relative roles of ER and AHR in manifesting the pharmacological actions and therapeutic efficacy of TAM and other SERMs.
| S-EPMC2802893 | biostudies-literature
Project description:Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.
| S-EPMC3955358 | biostudies-literature