Research Resource: Rapid Recruitment of Temporally Distinct Vascular Gene Sets by Estrogen

Abstract

Cardiovascular disease is the leading cause of mortality for both men and women in developed countries. The sex steroid hormone estrogen is required for normal vascular physiology. Estrogen functions by binding to intracellular estrogen receptors (ER), ERalpha and ERbeta, ligand-activated transcription factors that are expressed in both vascular endothelial and smooth muscle cells. We recently demonstrated that long-term (8 d) estrogen treatment in vivo in mice recruits distinct vascular gene sets mediated by ERalpha and ERbeta and that the promoters from these gene sets are enriched for binding sites of specific transcription factors, leading to the hypothesis that estrogen initiates a cascade of early transcriptional events that modulate gene expression in the vasculature. Here we test this hypothesis using gene expression profiling to examine initial transcriptional events (2-8 h) mediated by estrogen in blood vessels. Our data reveal that 1) estrogen regulates temporally distinct cascades of vascular gene expression, 2) initially, estrogen-mediated vascular gene repression predominates, 3) the earliest estrogen-recruited gene program is enriched in vascular transcription factors that can interact with binding sites present in estrogen-regulated vascular genes recruited subsequently, and 4) estrogen-regulated genes recruited next have specific functions, including lipid metabolism and cellular growth and proliferation that are potentially important for estrogen's known vascular functions. In summary, estrogen directly and rapidly recruits specific transcriptional factors that then propagate distinct cascades of gene expression. These data define the temporal recruitment of specific vascular genes by estrogen and enable further analysis of the mechanisms by which estrogen directly regulates vascular function.

Estrogen Receptor–Mediated Regulation of MicroRNA Inhibits Proliferation of Vascular Smooth Muscle Cells

“…9, 16, 17 However, we recently demonstrated that many ER-regulated genes in the mouse aorta are down-regulated rather than up-regulated. 18, 19 Similar findings have been demonstrated in other, non-vascular cells as well. 20 The molecular mechanisms by which ERs repress gene expression are currently unknown.…”

“…19 3 aortas were pooled for each assay. For each treatment group, miR arrays and real-time PCR were performed in duplicate and quadruplicate, respectively, as described below.…”

Placental growth factor mediates aldosterone-dependent vascular injury in mice

“…Total vessel RNA was isolated and reverse transcribed, and QRT-PCR was carried out as previously described (56) with primers specific for PGF, Flt1, or Flk1 (see Supplemental Table 2 for primer sequences). The CT value for each gene was normalized to that of β2-microglobulin (for mouse tissues) or GAPDH (for human tissues) and expressed as fold change in treated samples compared with normal or vehicle-treated samples as previously described (56). Between 3 and 6 mouse or human vessels were used for each condition.…”

Research Resource: Aorta- and Liver-Specific ERα-Binding Patterns and Gene Regulation by Estrogen

“…qPCR was performed using SYBR Green Master Mix (QIAGEN), with each PCR performed in triplicate. Gene expression levels were normalized to ␤ 2 -microglobulin (a well-established normalization control for E 2 -regulated gene expression in mouse tissues) (30,31) and ER␣ binding was normalized to an intergenic region with no detectable ER␣ binding in mouse tissues (Brown, M., and J. Cook, personal communication). We tested 11 genes that were called by RNA-Seq as at least 1.35-fold differentially regulated by E 2 between tissues ([AoE Ϫ AoV] Ϫ [LiE Ϫ LiV]) by qRT-PCR (not all of which also met the P Ͻ .05 and fold change Ͼ1.5 cutoffs for inclusion in the lists of regulated genes by RNA-Seq).…”

“…We examined gene regulation by estrogen in mouse aorta and liver using an established ex vivo treatment model (30). Female C57/BL6 mice, aged 6 to 7 weeks, were ovariectomized to remove the principal source of endogenous circulating estrogen (9).…”