17β-Estradiol Modulates Gene Expression in the Female Mouse Cerebral Cortex

<div><p>17β-estradiol (E2) plays critical roles in a number of target tissues including the mammary gland, reproductive tract, bone, and brain. Although it is clear that E2 reduces inflammation and ischemia-induced damage in the cerebral cortex, the molecular mechanisms mediating the effects of E2 in this brain region are lacking. Thus, we examined the cortical transcriptome using a mouse model system. Female adult mice were ovariectomized and implanted with silastic tubing containing oil or E2. After 7 days, the cerebral cortices were dissected and RNA was isolated and analyzed using RNA-sequencing. Analysis of the transcriptomes of control and E2-treated animals revealed that E2 treatment significantly altered the transcript levels of 88 genes. These genes were associated with long term synaptic potentiation, myelination, phosphoprotein phosphatase activity, mitogen activated protein kinase, and phosphatidylinositol 3-kinase signaling. E2 also altered the expression of genes linked to lipid synthesis and metabolism, vasoconstriction and vasodilation, cell-cell communication, and histone modification. These results demonstrate the far-reaching and diverse effects of E2 in the cerebral cortex and provide valuable insight to begin to understand cortical processes that may fluctuate in a dynamic hormonal environment.</p></div

E2-regulated genes with the most increased expression.

<p>E2-regulated genes with the most increased expression.</p

E2 increases pERK protein levels.

<p>(A)Western blot analysis was used to monitor pERK and total ERK levels in the cortices of mice that had been treated with oil or E2 for 7 days. (B) pERK values were normalized to total ERK and are displayed as the normalized fold change ± SEM. The Student’s t-test was used to detect significant differences in oil- and E2- treated animals (*<i>p</i><0.05). The number of animals in each treatment group is indicated at the base of each bar.</p

Validation of transcripts altered by E2 treatment.

<p>(A) Quantitative real-time PCR was conducted with gene-specific primers. The normalized fold change ± SEM was calculated using the delta-delta Ct method with Rpl7 as a control gene. The Student’s t-test was used to detect significant differences in oil- and E2- treated animals (4 animals/treatment, *<i>p</i><0.05).</p

Networks of E2-regulated genes.

<p>ClueGO analysis classified the 88 E2-regulated genes into 6 networks. White nodes indicate that a biological process is associated with two networks. Node size indicates the statistical significance of the biological process represented. Thus, larger nodes indicate greater statistical significance.</p

E2-responsive networks and associated genes.

<p>E2-responsive networks and associated genes.</p

88 E2-responsive genes in the cerebral cortex.

<p>88 E2-responsive genes in the cerebral cortex.</p

E2-regulated genes with the most decreased expression.

<p>E2-regulated genes with the most decreased expression.</p

IGFBPs, ECM proteins, and other proteins detected in the CM of BC and MCF-10A cells.

<p>A-C. The number of SCs identified in the CM of each cell line is indicated. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. NSAF data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158296#pone.0158296.s003" target="_blank">S3 Table</a>. D. BC gene expression data was extracted from TCGA and boxplots were created for luminal A (pink) and TNBC (blue) tumors. Expression values are log2 normalized, tumor matched normal, with normal mammary tissue expression set to 0. (Abbreviations: CM = conditioned medium, BC = breast cancer, SC = spectral counts, TCGA = The Cancer Genome Atlas, TNBC = triple negative breast cancer)</p

Putative exosomal proteins detected in the CM of BC and MCF-10A cells.

<p>A. The number of SCs identified in the CM of each cell line is indicated. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. NSAF data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158296#pone.0158296.s003" target="_blank">S3 Table</a>. B. STRING analysis illustrates the numerous complex interactions possible among the putative exosomal proteins. Thicker lines reflect a higher confidence score. (Abbreviations: CM = conditioned medium, BC = breast cancer, SC = spectral counts)</p