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Estrogen-regulated gene expression in human osteosarcoma cells stably transfected with estrogen receptor alpha or beta

ABSTRACT: Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable to those in osteoblasts, and we characterized their response to estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the ca. 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48 h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE1153 | GEO | 2004/04/10

SECONDARY ACCESSION(S): PRJNA87283

REPOSITORIES: GEO

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Project description:In addition to their role in the development and function of the reproductive system, estrogens have significant anti-inflammatory properties. Although both estrogen receptors (ERs) can mediate anti-inflammatory actions, ERbeta is a more desirable therapeutic target because ERalpha mediates the proliferative effects of estrogens on the mammary gland and uterus. In fact, selective ERbeta agonists have beneficial effects in preclinical models involving inflammation without causing growth-promoting effects on the uterus or mammary gland. However, their mechanism of action is unclear. The purpose of this study was to use microarray analysis to determine whether ERbeta-selective compounds produce their anti-inflammatory effects by repressing transcription of proinflammatory genes. We identified 49 genes that were activated by TNF-alpha in human osteosarcoma U2OS cells expressing ERbeta. Estradiol treatment significantly reduced the activation by TNF-alpha on 18 genes via ERbeta or ERalpha. Most repressed genes were inflammatory genes, such as TNF-alpha, IL-6, and CSF2. Three ERbeta-selective compounds, ERB-041, WAY-202196, and WAY-214156, repressed the expression of these and other inflammatory genes. ERB-041 was the most ERbeta-selective compound, whereas WAY-202196 and WAY-214156 were the most potent. The ERbeta-selective compounds repressed inflammatory genes by recruiting the coactivator, SRC-2. ERB-041 also repressed cytokine genes in PBMCs, demonstrating that ERbeta-selective estrogens have anti-inflammatory properties in immune cells. Our study suggests that the anti-inflammatory effects of ERB-041 and other ERbeta-selective estrogens in animal models are due to transcriptional repression of proinflammatory genes. These compounds might represent a new class of drugs to treat inflammatory disorders. Experiment Overall Design: Expression of ER in the U2OS-ERÃ cells was induced with doxycycline. The cells were treated in the absence or presence of 10 nM E2 for 18 h followed by incubation with TNF-{alpha} for 1 h. Total RNA were extracted, followed by cRNA labelling, and hybridization of fragmented samples to the U95Av2 arrays (n = 3 for untreated, n = 4 for TNF-{alpha}-treated, n = 4 for TNF-{alpha}- and E2-treated samples). The data were analyzed using the Microarray Suite Version 5.0 with the default parameters. The comparative data generated for each treated group were analyzed further in Microsoft Excel. TNF-{alpha}-induced genes were selected for further analysis only if they had a 1.2 signal log ratio mean value (2.3-fold change) and were statistically significant (p < 0.05) in at least three separate experiments.

Project description:Estrogen plays an important role in the regulation of vascular tone and in the pathophysiology of cardiovascular disease. Physiological effects of estrogen are mediated through estrogen receptors alpha (ERalpha) and beta (ERbeta), which are both expressed in vascular smooth muscle and endothelial cells. However, the molecular pathways mediating estrogen effects in blood vessels are not well defined. We have performed gene expression profiling in the mouse aorta to identify comprehensive gene sets the expression of which is regulated by long-term (1 wk) estrogen treatment. The ER subtype dependence of the alterations in gene expression was characterized by parallel gene expression profiling experiments in ERalpha-deficient [ERalpha knockout (ERalphaKO)] and ERbeta-deficient (ERbetaKO) mice. Importantly, these data revealed that ERalpha- and ERbeta-dependent pathways regulate distinct and largely nonoverlapping sets of genes. Whereas ERalpha is essential for most of the estrogen-mediated increase in gene expression in wild-type aortas, ERbeta mediates the large majority of estrogen-mediated decreases in gene expression. Biological functions of the estrogen-regulated genes include extracellular matrix synthesis, in addition to electron transport in the mitochondrion and reactive oxygen species pathways. Of note, the estrogen/ERbeta pathway mediates down-regulation of mRNAs for nuclear-encoded subunits in each of the major complexes of the mitochondrial respiratory chain. Several estrogen-regulated genes also encode transcription factors. Overall, these findings provide a foundation for understanding the molecular basis for estrogen effects on vasculature gene expression. Experiment Overall Design: Six estrogen receptor alpha knockout (ERaKO) and six estrogen receptor beta knockout (ERbKO) mice and ten of their wild-type littermates (all female, 2.5-4.5 months of age) were ovarioectomized. Half the mice from each genotype were implanted with 17beta-estradiol pellets, the other half with placebo pellets. After 7-8 days of estrogen/placebo treatment, aortas were harvested, total RNAs were purified for Affymetrix GeneChip microarray analysis, without pooling. This experiment consists of 6 groups with 3 (ERaKO and ERbKO) or 5 (WT) biological replicates per group, for a total of 22 samples.

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Estrogen-regulated gene expression in human osteosarcoma cells stably transfected with estrogen receptor alpha or beta

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