Endogenous moR-21 downregulates Txndc5 expression.

<p>(a) Txndc5 protein abundance in scrambled or moR-21 mimetic and scrambled or anti-moR-21 treated cells. Representative western blots are presented. Densitometry analysis for western blots is shown in the lower panel. Data are from at least 4 independent experiments and presented as fold change, compared with scrambled mimetic and scrambled anti-moR treated cells. (b) Upper panel: the moR-21 sponge was designed with bulged binding sites to avoid endonucleolytic cleavage by Ago2. Lower panel: the moR-21 sponge was constructed by inserting 7 copies of bulged moR-21 binding sites into the 3’UTR of a destabilized GFP reporter gene. (c) qRT-PCR analysis of Txndc5 mRNA in control or moR-21 sponge transfected HEK293 cells. Gapdh was used as an internal control for RT-qPCR. (d) immunoblot analysis of Txndc5 protein abundance in control or moR-21 sponge transfected HEK293 cells. Data are from 4 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

Endogenous moR-21 downregulates Txndc5 expression.

<p>(a) Txndc5 protein abundance in scrambled or moR-21 mimetic and scrambled or anti-moR-21 treated cells. Representative western blots are presented. Densitometry analysis for western blots is shown in the lower panel. Data are from at least 4 independent experiments and presented as fold change, compared with scrambled mimetic and scrambled anti-moR treated cells. (b) Upper panel: the moR-21 sponge was designed with bulged binding sites to avoid endonucleolytic cleavage by Ago2. Lower panel: the moR-21 sponge was constructed by inserting 7 copies of bulged moR-21 binding sites into the 3’UTR of a destabilized GFP reporter gene. (c) qRT-PCR analysis of Txndc5 mRNA in control or moR-21 sponge transfected HEK293 cells. Gapdh was used as an internal control for RT-qPCR. (d) immunoblot analysis of Txndc5 protein abundance in control or moR-21 sponge transfected HEK293 cells. Data are from 4 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

MoR-21L plays a functional role in VSMC.

<p>(a) Location of moRs and miRs sequences on the predicted secondary structure surrounding the pre-miR-21 hairpin. The RNA structure prediction software mFold was used to predict pre-miR-21 secondary structure. (b) Increased expression of moR-21 and miR-21 in injured mouse carotid artery. Data are shown as mean ± SEM and are from 3 independent experiments. (c) Abundance of moR-21 and miR-21 in VSMC cultured under different conditions. VSMC were cultured in serum free medium (SFM) and medium containing 10% fetal bovine serum (FBS) or PDGF. BSA is the control for PDGF. Data are shown as mean ± SEM and are from 4 independent experiments. (d) Effects of over-expression of moR-21, miR-21, and scrambled mimetics on VSMC proliferation. VSMC were transfected with 20nM scrambled control, moR-21, and or miR-21 mimetics. Cell proliferation was measured at 0, 1, 2, and 3 days using Cell TiterGlo. Data are presented as relative proliferation, compared with scrambled mimetic-transfected cells on day 0. Data are shown as mean ± SEM and are from 5 independent experiments. <i>P</i> values were determined by one way repeated measure ANOVA. NS: nonsignificant, *: <i>P</i><0.05, ***: <i>P</i><0.001.</p

Ago2 is required for moR-21-mediated gene repression.

<p>(a) Immunoblot analysis showing the effects of knocking-down Ago1-4 on Igfr1 and Txndc5 protein abundance in moR-21 over-expressing cells. Scr: transfection control with scrambled siRNA or small RNA mimetic. Gapdh: loading control. Lower bar graphs show densitometry results in line with the representative blots. Data are from 3–5 independent experiments and presented as fold change. Values are mean ± SEM. <b>b,c</b> RIP analysis of HEK293 cell lysates to measure the interaction of Ago2 with endogenous miR-21 or moR-21 (b), and Txndc5 (c). Normal mouse IgG served as IP control. MoR-21, miR-21, or Txndc5 abundance was quantified using RT-qPCR and represented as fold enrichment in Ago2 RIP compared with IgG RIP. Data are from 4 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i> <0.001.</p

moR-21 plays a role in gene regulation and has a different target gene set from miR-21.

<p>(a) Differentially expressed transcripts in moR-21 over-expressed VSMC compared with scrambled control-treated cells. In moR-21-treated cells, 460 and 378 transcripts were down- or up-regulated, respectively. Fold change cutoff is 1.3 and adjusted <i>P</i>< 0.002. Right panel: a differential expression heatmap for all 838 genes that were significantly regulated by moR-21 showing the log base 2 expression value for each sample minus the average log base 2 expression value for all samples (three control and three moR-21 treated). (b) Comparison between miR-21 and moR-21 targets predicted by TargetScan and their regulation by moR-21, as detected by microarray in VSMC. (c) Comparison of molecular pathways that predicted miR-21 and moR-21 targets participate in. The analysis was performed using IPA with a <i>P</i>-value cutoff < 0.05. d, Pik3r1, Igf1r, and Txndc5 protein abundance in scrambled control, moR-21, or miR-21 mimetic treated mAoSMC cells. Representative western blots are presented. Densitometry analysis for western blots is shown at the bottom. e. Conserved moR-21 binding sites in the 3’UTR of Txndc-5 and Igf1r. Data are from at least 4 independent experiments and presented as fold change. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

“Seed” region plays a key role in moR-21-mediated gene down-regulation.

<p>(a) Target prediction analysis for all potential “seed” sequences (highlighted in red). Numbers of predicted targets that were significantly down-regulated, up-regulated or unchanged in the microarray are represented by blue, red, and grey bars, respectively. (b) Overlap among down-regulated targets predicted by the first 3 “seed” sequences. (c) The common region of the first 3 “seed” sequences is GUACC. Each moR-21 mutant (Mut1 and Mut2) carries 2 nucleotide substitutions in the common region, which are highlighted in red. (d) Effects of wild-type and mutated moR-21 mimetics on the mRNA levels of the 11 targets genes predicted by 2 or 3 potential “seed” sequences. Each gene expression level was calculated as relative fold change compared to the scrambled control-treated sample. B2M was used as an internal control for RT-qPCR. Data are shown as mean ± SEM and are from at least 5 independent experiments. (e) Txndc5 protein abundance in scrambled control, wild type moR-21, and mutated moR-21 treated cells. Representative western blot is presented. Densitometry analysis for western blots is shown at the bottom. Data are shown as mean ± SEM and are from at least 3 independent experiments. (f) (g) Schematic presentation of Txndc5 3’UTR luciferase reporter and luciferase assay. A perfect match between the “seed” region in moR-21 and the “seed match” region in Txndc5 3’UTR is represented by short vertical lines. Nucleotide substitutions in moR-21 “seed” region (f) and Txndc5 3’UTR “seed match” region (g) are highlighted in red. Luciferase activity was normalized to beta-gal activity. Data are presented as fold change, compared with scrambled control-treated cells. Values shown are mean ± SEM of at least 3 independent experiments. The significance of differences between different treatments was determined by one-way analysis of variance (ANOVA), followed by Tukey’s test (b) or Student’s <i>t</i>-test (c), and Student’s t-test (d,e). NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells

<div><p>Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.</p></div

Distinct TF binding site enrichments in hEC genes regulated by E2/ERα rapid signaling.

<p><b>(A)</b> Fold enrichments, and median adjusted p. values for every TF represented by more than one significantly-enriched TFBS matrix (adjusted p. value < 0.05 and enrichment over background, non-regulated gene promoters > 1.15), and which showed at least a 1.05-fold enrichment for all its cognate matrices. <b>(B & C)</b> Plots of the average frequency of matches to the significantly enriched CEBP and SRF matrices versus the TSSes of WT hEC E2 up-regulated genes, relative to the background from unregulated gene promoters.</p

KRR mutant ERα in stable EC cell lines activates genomic but not rapid signaling.

<p><b>(A)</b> Western blot for ERα or GAPDH (as an internal control) in Ctrl, WT or KRR stable Eahy926 cell lines. All WT or KRR clones had similar levels of ERα expression, while all Ctrl hEC clones had no detectable ERα expression (DNS). <b>(B-E)</b> WT or KRR hECs were treated +E2 or +Veh for 20 mins, followed by Western blotting for total versus phosphorylated active forms of endogenous Akt <b>(C)</b>, ERK <b>(D)</b> or eNOS <b>(E)</b>, as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152807#pone.0152807.g002" target="_blank">Fig 2A–2D</a>, with representative images in <b>(B)</b>. <b>(F)</b> WT or KRR hECs were transfected with ERE-luc & β-gal reporter plasmids, as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152807#pone.0152807.g002" target="_blank">Fig 2E</a>. *: p. <0.05. All WT or KRR clones had similar levels of ERE-binding genomic ERα function, measured as E2-induced luciferase expression in this assay, while all Ctrl hEC clones had no significant E2-induced luciferase activity (DNS).</p

Rapid signaling through ERα is required for E2-dependent EC proliferation & migration.

<p><b>(A)</b> Relative cell counts for stable Ctrl, WT or KRR hEC cell lines +/- 10nM E2, normalized to counts +Veh. <b>(B)</b> A “scratch” wound was made with a pipette tip on Control, WT or KRR hECs at near confluence, and the number of cells migrating into the scratch after 48 hours counted. Top: Quantitation of migration data. Bottom: representative images of cells after 48 hr migration. Dotted lines indicate the borders of the scratch at time 0. Data is normalized to +Veh for each cell line. *: different from WT+Veh, p. <0.05, #: different from KRR+E2, p. <0.05.</p