Estradiol suppresses NF-kappa B activation through coordinated regulation of let-7a and miR-125b in primary human macrophages - PubMed (original) (raw)

Estradiol suppresses NF-kappa B activation through coordinated regulation of let-7a and miR-125b in primary human macrophages

Amy J Murphy et al. J Immunol. 2010.

Abstract

Previous findings suggest that 17beta-estradiol (estradiol) has a suppressive effect on TNF-alpha, but the mechanism by which estradiol regulates TNF-alpha expression in primary human macrophages is unknown. In this article, we demonstrate that pretreatment of human macrophages with estradiol attenuates LPS-induced TNF-alpha expression through the suppression of NF-kappaB activation. Furthermore, we show that activation of macrophages with LPS decreases the expression of kappaB-Ras2, an inhibitor of NF-kappaB signaling. Estradiol pretreatment abrogates this decrease, leading to the enhanced expression of kappaB-Ras2 with LPS stimulation. Additionally, we identified two microRNAs, let-7a and miR-125b, which target the kappaB-Ras2 3' untranslated region (UTR). LPS induces let-7a and inhibits miR-125b expression in human macrophages, and pretreatment with estradiol abrogates these effects. 3'UTR reporter assays demonstrate that let-7a destabilizes the kappaB-Ras2 3'UTR, whereas miR-125b enhances its stability, resulting in decreased kappaB-Ras2 in response to LPS. Our data suggest that pretreatment with estradiol reverses this effect. We propose a novel mechanism for estradiol inhibition of LPS-induced NF-kappaB signaling in which kappaB-Ras2 expression is induced by estradiol via regulation of let-7a and miR-125b. These findings are significant in that they are the first to demonstrate that estradiol represses NF-kappaB activation through the induction of kappaB-Ras2, a key inhibitor of NF-kappaB signaling.

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Figures

Figure 1

Figure 1. Estradiol attenuates LPS-induced TNF-α

(A) ELISA analysis of TNF-α levels in culture supernatants from macrophages treated with estradiol or ethanol control for 24 hours prior to stimulation with LPS (10 ng/ml) for 12 hours. Data are presented as percent of TNF-α produced by LPS-stimulated cells not receiving estradiol treatment. (B) Total RNA was extracted from macrophages treated with or without estradiol (100 nM) followed by stimulation with LPS for 5 hours. TNF-α mRNA was measured by TaqMan real-time PCR. (C) Macrophages were treated with the estrogen receptor antagonist ICI 182,780 (1 μM) for 1 hour prior to estradiol treatment. Hormone and LPS treatments were the same as in A. TNF-α levels were measured by ELISA and are presented as percent of control treated cells stimulated with LPS. *p<0.05 vs. control cells stimulated with LPS.

Figure 2

Figure 2. Estradiol inhibits NF-κB signaling

(A) Cells were treated with NF-κB activation inhibitor (10 μM) for 1 hour followed by LPS stimulation for 5 hours. TNF-α levels in culture supernatants were measured by ELISA. N.D., not detected. (B) Whole cell lysates were prepared from macrophages pre-treated with estradiol and then stimulated with LPS for 1 hour. IKKβ, IκBα and phospho-IκBα levels were measured by immunoblot and GAPDH served as a loading control. (C) Cells were treated as in B, and nuclear lysates were prepared and analyzed for p65 by ELISA. Data are normalized to control treated, unstimulated lev28ls. *p<0.01 vs. LPS stimulation alone. **p<0.05 vs. control treated and unstimulated.

Figure 3

Figure 3. κB-Ras2 is up-regulated by estradiol

(A) Total RNA was extracted from macrophages treated with LPS at indicated times. κB-Ras2 mRNA was measured by TaqMan real-time PCR. Values are relative to unstimulated controls. (B) Immunoblot analysis of κB-Ras2 protein levels in response to LPS. (C–D) mRNA and protein levels, respectively, of κB-Ras2. Macrophages were treated with estradiol prior to LPS stimulation for 8 hours (C) or 12–24 hours (D). Data shown are representative of 4 experiments. *p< 0.05 vs. unstimulated control

Figure 4

Figure 4. Let-7a and miR-125b are regulated by LPS

(A) Total RNA was extracted from macrophages using Qiagen miRNeasy kits. Let-7a and miR-125b expression were measured using Taqman miRNA assay system. Data are normalized to U6 expression levels. (B) let-7a and (C) miR-125b expression levels in response to LPS stimulation. Data were normalized to U6 and are presented as relative to unstimulated levels. (D–E) Macrophages from a representative donor were treated with NF-κB activation inhibitor (10 μM) for 1 hour prior to LPS stimulation for (D) 12 hours or (E) 3 hours. *p<0.05 vs. unstimulated controls.

Figure 5

Figure 5. Estradiol inhibits LPS effect on let-7a and miR-125b

(A) let-7a levels in macrophages pre-treated with estradiol and stimulated with LPS for 12 hours. (B) miR-125b expression in cells treated with estradiol and stimulated with LPS for 3 hours. *p<0.05 vs. unstimulated controls.

Figure 6

Figure 6. let-7a and miR-125b directly target kB-Ras2 3′UTR

(A) Alignment of let-7a and miR-125b with κB-Ras2 3′UTR. Solid lines indicate Watson-Crick base pairs. Dotted line indicates GU wobble pairs. The gray background denotes the seed binding region. (B) Secondary structure as predicted by RNAHybrid with κB-Ras2 3′UTR shown in black and miRNA in gray. (C) RAW 264.7 cells were transfected with luciferase expression vector containing the κB-Ras2 3′UTR or the control vector and synthetic pre-miR-125b, pre-let-7a or a negative control pre-miRNA. The cells were also transfected with a vector containing Renilla luciferase to serve as a transfection efficiency control. After 48 hours, cells were lysed and analyzed for luciferase expression using a dual-luciferase assay system. (D) PMA-differentiated U937 cells were transfected with pre-miR-125b, pre-let-7a or pre-miRNA negative control and analyzed for κB-Ras2 expression after 48 hours. *p<0.05 vs. negative control miRNA.

Figure 7

Figure 7. Proposed mechanism of estradiol regulation of NF-κB signaling in primary human macrophages

(A) LPS binding to TLR4 induces expression of let-7a and decreases expression of miR-125b, leading to decreased expression of κB-Ras2 to enable complete activation of NF-κB signaling and TNF-α expression. (B) When macrophages are treated with estradiol prior to LPS stimulation, changes in expression of let-7a and miR-125b in response to 29LPS are abrogated. This results in up-regulation of κB-Ras2 and inhibition of NF-κB signaling, culminating in decreased expression of TNF-α.

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