Project description:Mouse BAL alveolar macrophages from pregnant E14 and control dams are compared to RAW cells cultured with estradiol to determine shared gene expression change patterns induced by pregnancy and estrogen BAL cells from intact pregnant and control mice were directly processed for RNA extraction. RAW cells were cultured overnight with estradiol or vehicle, then RNA was exctracted.
Project description:Selective estrogen receptor modulators (SERMs) are potentially useful in treating various endometrial disorders, including endometrial cancer, as they block some of the detrimental effects of estrogen. It remains unclear whether each SERM regulates a unique subset of genes and, if so, whether the combination of a SERM and 17beta-estradiol has an additive or synergistic effect on gene expression. We performed microarray analysis with Affymetrix Mouse Genome 430 2.0 short oligomer arrays to determine gene expression changes in uteri of ovariectomized mice treated with estradiol (low and high dose), methyl-piperidino-pyrazole (MPP), ICI 182 780, raloxifene, and combinations of high dose of estradiol with one of the SERM and dimethyl sulfoxide (DMSO) vehicle control. The nine treatments clustered into two groups, with MPP, raloxifene, and high dose of estradiol in one, and low dose of estradiol, ICI + estradiol, ICI, MPP + estradiol, and raloxifene + estradiol in the second group. Surprisingly, combining a high dose of estradiol with a SERM markedly increased (P<0.02) the number of regulated genes compared with each individual treatment. Analysis of expression for selected genes in uteri of estradiol and SERM-treated mice by quantitative (Q)RT-PCR generally supported the microarray results. For some cancer-associated genes, including Klk1, Ihh, Cdc45l, and Cdca8, administration of MPP or raloxifene with estradiol resulted in greater expression than estradiol alone (P<0.05). By contrast, ICI 182 780 suppressed more genes governing DNA replication compared with MPP and raloxifene treatments. Therefore, ICI 182 780 might be superior to MPP and raloxifene to treat estrogen-induced endometrial cancer in women.
Project description:BACKGROUND:Recent studies have highlighted the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior. Herein we report preclinical studies designed to characterize the effects of a novel JAK/STAT inhibitor on plantar flexor skeletal muscle function, morphology, and satellite cell content. METHODS:The compound, SGI-1252, was administered orally (400mg/kg) in a 10% dextrose solution to wild type mice (n = 6) 3 times per week for 8 weeks. A control group (n = 6) received only the dextrose solution. RESULTS:SGI-1252 was well tolerated, as animals displayed similar weight gain over the 8-week treatment period. Following treatment, fatigue in the gastrocnemius-soleus-plantaris complex was greater in the SGI-1252 mice during a 300 second tetanic contraction bout (p = 0.035), though both the rate of fatigue and maximal force production were similar. SGI-1252 treated mice had increased type II myofiber cross-sectional area (1434.8 ± 225.4 vs 1754.7 ± 138.5 ?m2), along with an increase in wet muscle mass (125.45 ± 5.46 vs 139.6 ± 12.34 mg, p = 0.032) of the gastrocnemius relative to vehicle treated mice. SGI-1252 treatment reduced gastrocnemius STAT3 phosphorylation 53% (94.79 ± 45.9 vs 44.5 ± 6.1 MFI) and significantly increased the concentration of Pax7+ satellite cells (2589.2 ± 105.5 vs 2859.4 ± 177.5 SC/mm3) in the gastrocnemius. SGI-1252 treatment suppressed MyoD (p = 0.013) and Myogenin (p<0.0001) expression in human primary myoblasts, resulting in reduced myogenic differentiation (p = 0.039). CONCLUSIONS:Orally delivered SGI-1252 was well tolerated, attenuates skeletal muscle STAT3 activity, and increases satellite cell content in mouse gastrocnemius muscle, likely by inhibiting myogenic progression.
Project description:This study aimed to assess the in vivo effects of estradiol treatment on arterial gene expression in atherosclerotic postmenopausal female monkeys.Eight ovariectomized cynomolgus monkeys were fed atherogenic diets for 6.5 years. The left iliac artery was biopsied before randomization to the estradiol group (human equivalent dose of 1 mg/d, n = 4) or the vehicle group (n = 4) for 8 months. The right iliac artery was obtained at necropsy. Transcriptional profiles in pretreatment versus posttreatment iliac arteries were compared to assess the responses of atherosclerotic arteries to estradiol.Iliac artery plaque size did not differ between the estradiol group and the placebo group at baseline or during the treatment period. Nevertheless, estradiol treatment was associated with increased expression of 106 genes and decreased expression of 26 genes in the iliac arteries. Estradiol treatment increased the expression of extracellular matrix genes, including the ?1 chain of type I collagen, the ?2 chain of type VI collagen, and fibulin 2, suggestive of an increase in the proportion or phenotype of smooth muscles or fibroblasts in lesions. Also increased were components of the insulin-like growth factor pathway (insulin-like growth factor 1, insulin-like growth factor binding protein 4, and insulin-like growth factor binding protein 5) and the Wnt signaling pathway (secreted frizzled-related protein 2, secreted frizzled-related protein 4, low-density lipoprotein receptor-related protein 6, and Wnt1-inducible signaling pathway protein 2).Estradiol treatment of monkeys with established atherosclerosis affected iliac artery gene expression, suggesting changes in the cellular composition of lesions. Moreover, it is probable that the presence of atherosclerotic plaque affected the gene expression responses of arteries to estrogen.
Project description:During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mER) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mER agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STX-treated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative real-time PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ERalpha and mER-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mER activation.
Project description:BACKGROUND: The mouse skeletal muscle is composed of four distinct fiber types that differ in contractile function, number of mitochondria and metabolism. Every muscle type has a specific composition and distribution of the four fiber types. To find novel genes involved in specifying muscle types, we used microarray analysis to compare the gastrocnemius with the quadriceps from mice fed a low fat diet (LFD) or high fat diet (HFD) for 8 weeks. Additional qPCR analysis were performed in the gastrocnemius, quadriceps and soleus muscle from mice fed an LFD or HFD for 20 weeks. RESULTS: In mice fed the 8-week LFD 162 genes were differentially expressed in the gastrocnemius vs. the quadriceps. Genes with the strongest differences in expression were markers for oxidative fiber types (e.g. Tnni1) and genes which are known to be involved in embryogenesis (Dkk3, Hoxd8,Hoxd9 and Tbx1). Also Dkk2, Hoxa5, Hoxa10, Hoxc9, Hoxc10, Hoxc6 and Tbx15 were detectably, but not differentially expressed in adult muscle tissue. Expression of differentially expressed genes was not influenced by an 8-week or 20-week HFD. Comparing gastrocnemius, quadriceps and soleus, expression of Hoxd8 and Hoxd9 was not related with expression of markers for the four different fiber types. We found that the expression of both Hoxd8 and Hoxd9 was much higher in the gastrocnemius than in the quadriceps or soleus, whereas the expression of Dkk3 was high in quadriceps, but low in both gastrocnemius and soleus. Finally, expression of Tbx1 was high in quadriceps, intermediate in soleus and low in gastrocnemius. CONCLUSIONS: We found that genes from the Dkk family, Hox family and Tbx family are detectably expressed in adult mouse muscle. Interestingly, expression of Dkk3, Hoxd8, Hoxd9 and Tbx1 was highly different between gastrocnemius, quadriceps and soleus. In fact, every muscle type showed a unique combination of expression of these four genes which was not influenced by diet. Altogether, we conclude that genes important for embryogenesis identify mouse muscle types in a diet-independent and fiber type-unrelated manner.
Project description:Estrogens are neuroprotective and, thus, potentially useful for the therapy of Alzheimer's disease; however, clinical use of hormone therapy remains controversial due to adverse peripheral effects. The goal of this study was to investigate the benefits of treatment with 10?,17?-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective prodrug of 17?-estradiol, in comparison with the parent hormone using APPswe/PS1dE9 double transgenic mice to model the pathology of the disease. Ovariectomized and intact females were continuously treated with vehicle, 17?-estradiol, or DHED via subcutaneous osmotic pumps from 6 to 8months of age. We confirmed that this prolonged treatment with DHED did not stimulate uterine tissue, whereas 17?-estradiol treatment increased uterine weight. Amyloid precursor protein decreased in both treatment groups of intact, but not in ovariectomized double transgenic females in which ovariectomy already decreased the expression of this protein significantly. However, reduced brain amyloid-? peptide levels could be observed for both treatments. Consequently, double-transgenic ovariectomized and intact mice had higher cognitive performance compared to untreated control animals in response to both estradiol and DHED administrations. Overall, the tested brain-selective 17?-estradiol prodrug proved to be an effective early-stage intervention in an Alzheimer's disease-relevant mouse model without showing systemic impact and, thus, warrants further evaluation as a potential therapeutic candidate.
Project description:To test if estrogen promotes carcinogenesis in vitro and in a genetic mouse model of ovarian cancer and whether its effects can be inhibited by a novel selective estrogen receptor modulator (SERM), bazedoxifene.Bazedoxifene was synthesized and it was confirmed that the drug abrogated the uterine stimulatory effect of 17?-estradiol in mice. To determine if hormones alter tumorigenesis in vivo LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with vehicle control, 17?-estradiol or bazedoxifene. Hormone receptor status of a cell line established from LSL-K-ras(G12D/+)Pten(loxP/loxP) mouse ovarian tumors was characterized using Western blotting and immunohistochemistry. The cell line was treated with hormones and invasion assays were performed using Boyden chambers and proliferation was assessed using MTT assays.In vitro 17?-estradiol increased both the invasion and proliferation of ovarian cancer cells and bazedoxifene reversed these effects. However, in the genetic mouse model neither treatment with 17?-estradiol nor bazedoxifene changed mean tumor burden when compared to treatment with placebo. The mice in all treatment groups had similar tumor incidence, metastatic nodules and ascites.While 17?-estradiol increases the invasion and proliferation of ovarian cancer cells, these effects do not translate into increased tumor burden in a genetic mouse model of endometrioid ovarian cancer. Likewise, while the SERM reversed the detrimental effects of estrogen in vitro, there was no change in tumor burden in mice treated with bazedoxifene. These findings demonstrate the complex interplay between hormones and ovarian carcinogenesis.
Project description:The cardiovascular effects of estrogen are mediated in part by augmenting the function of endothelial nitric oxide synthase. Endothelial nitric oxide synthase activity is dependent on many cofactors including Ca(2+). Hence, we investigated the effect of chronic 17 beta-estradiol treatment on the intracellular Ca(2+) concentration and endothelial nitric oxide synthase protein expression in the human endothelial cell line, EA.hy926, using spectrofluorometry and Western blot, respectively. Inhibiting the sarco(endo)plasmic reticulum Ca(2+) ATPase with thapsigargin caused an increase in the intracellular Ca(2+) concentration, which was higher in chronically 17 beta-estradiol-treated (1muM, 24h) cells loaded with Fura-2-acetoxymethyl ester compared to vehicle-treated cells, suggesting a higher endoplasmic reticulum Ca(2+) content in 17 beta-estradiol-treated cells. An enhanced Ca(2+) influx pathway in chronically 17 beta-estradiol-treated cells was also observed. In addition, 17 beta-estradiol-treated cells expressed higher levels of endothelial nitric oxide synthase protein in comparison to vehicle-treated cells. The chronic effect of 17 beta-estradiol on Ca(2+) homeostasis and endothelial nitric oxide synthase expression was attenuated with the nonselective estrogen receptor inhibitor, ICI 182,780 (10muM, 7alpha, 17beta-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl] estra-1,3,5(10)-triene-3,17-diol). Furthermore, analysis of the thapsigargin-evoked Ca(2+) response in chronically 17 beta-estradiol-treated estrogen receptor alpha-knockdown cells showed no significant difference in Ca(2+) response compared to vehicle-treated estrogen receptor alpha-knockdown cells, indicating that the regulation of Ca(2+) homeostasis by 17 beta-estradiol is mediated through an estrogen receptor alpha-dependent pathway. These data revealed an estrogen receptor alpha-dependent modulation of Ca(2+) homeostasis accompanying the enhancement of endothelial nitric oxide synthase expression in 17 beta-estradiol-treated human endothelial cells.