Previous findings suggest that 17β-estradiol (estradiol) has a suppressive effect on TNF-α, but the mechanism by which estradiol regulates TNF-α expression in primary human macrophages is unknown. In this article, we demonstrate that pretreatment of human macrophages with estradiol attenuates LPS-induced TNF-α expression through the suppression of NF-κB activation. Furthermore, we show that activation of macrophages with LPS decreases the expression of κB-Ras2, an inhibitor of NF-κB signaling. Estradiol pretreatment abrogates this decrease, leading to the enhanced expression of κB-Ras2 with LPS stimulation. Additionally, we identified two microRNAs, let-7a and miR-125b, which target the κB-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 κB-Ras2 3′UTR, whereas miR-125b enhances its stability, resulting in decreased κB-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-κB signaling in which κB-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-κB activation through the induction of κB-Ras2, a key inhibitor of NF-κB signaling.
To limit microbial pathogenesis, mammalian host cells rely on innate and adaptive immune mechanisms. The innate immune system deploys rapid antimicrobial responses to pathogenic challenge and simultaneously instructs the adaptive immune system regarding the nature and context of the infectious threat. As key phagocytes, macrophages provide early recognition of pathogens and a crucial bridge between innate and adaptive immunity. This is mediated through the detection of distinct molecules that are present on a broad diversity of microorganisms. These conserved products, termed pathogen-associated molecular patterns, are recognized by a repertoire of invariant pattern-recognition receptors, including the TLRs. TLR4, in association with accessory molecules MD-2 and CD14, is the signal transduction receptor for Gram-negative bacterial LPS. Engagement of TLR4 by LPS initiates a cascade of signaling events that culminates in the production of inflammatory cytokines, including TNF-α (1).
TNF-α is a potent proinflammatory cytokine produced by activated macrophages and has pleiotropic effects on immune cell survival, activation, and differentiation. During an infection, pathogen clearance is dependent on proper regulation of TNF-α, but aberrant expression of TNF-α can lead to significant morbidity and mortality (2). Deregulated TNF-α production has been correlated with disease activity in numerous autoimmune disorders, including rheumatoid arthritis and systemic lupus erythematosus, which are much more prevalent in women than in men (3). Intriguingly, serum TNF-α levels are lower in healthy premenopausal females compared with males and postmenopausal females (4), and TNF-α levels are attenuated in female patients during septic shock compared with males (5). Previous work suggested that 17β-estradiol (estradiol) has a suppressive effect on TNF-α production in cell lines and murine macrophages (6, 7), although no study has addressed the mechanism by which estradiol regulates TNF-α expression in primary human macrophages.
LPS-induced TNF-α expression is regulated by NF-κB activation. In unstimulated cells, the NF-κB transcription factors p65 and p50 are bound to IκB and are inactive. LPS binding to TLR4 leads to activation of the IκB kinase (IKK) complex that subsequently phosphorylates IκB, targeting it for degradation. The p65 and p50 NF-κB subunits are then free to translocate to the nucleus where they modulate the transcription of NF-κB–regulated genes.
Activation of NF-κB can be regulated through the binding of proteins that modulate IκB degradation. κB-Ras2 is a member of the Ras family of proteins that negatively regulates NF-κB signaling. κB-Ras2 binds directly to IκBα and inhibits IKK-dependent phosphorylation and subsequent degradation, thus inhibiting NF-κB signaling (8, 9). In this study, we demonstrate that pretreatment of human macrophages with estradiol attenuates LPS-induced TNF-α expression through the inhibition of NF-κB activation. Furthermore, we show that activation of macrophages with LPS inhibits the expression of κB-Ras2 but that pretreatment with estradiol abrogates this inhibition, leading to enhanced expression of κB-Ras2.
Because microRNAs (miRNAs) have been increasingly implicated in the regulation of immune function, we hypothesized that miRNAs might be involved in the mechanism by which estradiol regulates the expression of κB-Ras2. miRNAs are short ∼22-nt regulatory RNAs that bind to the 3′UTRs of target mRNAs and modulate stability and translation. Using an miRNA prediction-binding program (10), we identified let-7a and miR-125b as miRNAs predicted to interact with the κB-Ras2 3′UTR. Intriguingly, we now show that LPS induces let-7a and inhibits miR-125b expression in primary human macrophages and that pretreatment of these cells with estradiol abrogates the regulation of these miRNAs by LPS. Therefore, we propose a novel mechanism for estradiol inhibition of LPS-induced NF-κB signaling in which κB-Ras2 expression is induced by estradiol via the coordinated inhibition of let-7a and the induction of miR-125b. Our model for this mechanism is depicted in Fig. 7A and 7B. These findings are significant in that they are the first to demonstrate that estradiol represses NF-κB activation through the induction of κB-Ras2, a key inhibitor of NF-κB signaling.
Materials and Methods
Isolation and culture of human peripheral blood monocytes and macrophages
PBMCs were obtained by leukapheresis of normal, healthy premenopausal female donors following informed consent. Written consent was obtained from all subjects in accordance with the human experimentation guidelines established by Dartmouth College’s Committee for the Protection of Human Subjects, protocol #17011. To preclude confounding results associated with exogenous hormone use, individuals using hormonal contraceptives were excluded from these studies. Mononuclear cells were separated on Ficoll-Hypaque and enriched for monocytes using cold aggregation (11). Monocyte purity was >95% as determined by CD14 expression using flow cytometry (data not shown). To generate macrophages, monocytes were cultured in the presence of 10 μg/ml GM-CSF (PeproTech, Rocky Hill, NJ) for 7 d. Macrophages were cultured in complete HEPES-buffered RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10% FBS (Hyclone, Logan, UT) and 50 μg/ml gentamicin sulfate (Sigma-Aldrich, St. Louis, MO).
Estradiol, LPS, and inhibitor treatments
Forty-eight hours prior to hormone treatment, culture media was replaced with HEPES-buffered phenol red-free RPMI 1640 (Mediatech) supplemented with 10% charcoal dextran-stripped FBS (Hyclone) and 50 μg/ml gentamicin sulfate. All hormone treatments were performed using this media. Estradiol (Calbiochem, San Diego, CA) was resuspended in ethanol immediately prior to treatment. Cells were treated with 100 nM estradiol, unless otherwise indicated, or ethanol as a vehicle control. For LPS-stimulation experiments, cells were pretreated with estradiol for 24 h followed by the administration of 10 ng/ml ultrapure Escherichia coli LPS (Sigma-Aldrich) for 12 h, unless otherwise noted. Culture supernatants were analyzed for TNF-α production using the human TNF-α Quantikine ELISA kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. For estrogen receptor (ER)-inhibitor experiments, cells were treated with 1 μM ICI 182,780 (Tocris Bioscience, Ellisville, MO) for 1 h prior to hormone treatment. In previous studies, we determined that the incubation of macrophages with 1 μM of the ER antagonist ICI 182,780 optimally blocks the binding of estradiol (10−7 M) to the ER (12, 13). For NF-κB–inhibition experiments, macrophages were treated with 10 μM 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline (EMD Chemicals, Gibbstown, NJ) for 1 h prior to LPS stimulation. As we previously reported, none of the hormone or antagonist doses affected macrophage viability (12, 14).
Total RNA extraction and real-time PCR
Total RNA, including miRNA, was extracted from cells using the miRNeasy Mini kit with on-column DNase I treatment (Qiagen, Valencia, CA). RNA integrity and concentration were determined using the RNA6000 Nano LabChip kit (Agilent, Palo Alto, CA). For mRNA analysis, 500 ng RNA were reverse transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. miRNA reverse transcription was performed using the TaqMan microRNA Reverse Transcription Kit and miRNA-specific primers (Applied Biosystems, Foster City, CA). Real-time TaqMan PCR was used to quantify mRNA and miRNA expression using TaqMan Master Mix and validated primer/probe sets (Applied Biosystems). Amplification was carried out using the Applied Biosystems 7300 Real-Time PCR system. Threshold cycle number was determined using Opticon software. mRNA levels were normalized to β-actin, and miRNA levels were normalized to U6 expression using the equation 2−(Et−Rt), where Rt is the mean cycle threshold for the control gene, and Et is the mean threshold for the experimental gene. Cycling conditions for TaqMan PCR consisted of an initial incubation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Product accumulation was measured during the extension phase, and all samples were run in triplicate.
Preparation of lysates and immunoblot analysis
Whole-cell lysates were prepared using passive lysis buffer (Promega, Madison, WI). Lysates were analyzed for total protein concentration using a Micro BCA Protein Assay kit (Pierce, Rockford, IL). Twenty-five micrograms of each lysate were separated on 10% acrylamide gels and electrotransferred to nitrocellulose membrane in Tris-glycine buffer with 20% methanol. Membranes were blocked with 5% milk in 1× TBS and 0.1% Tween-20 for 1 h at room temperature. Blots were then probed with primary detection Abs for IκB kinaseβ (IKKβ; AbCam, Cambridge, MA), IκBα (Cell Signaling Technology, Beverly, MA), phospho-IκBα (Cell Signaling), or κB-Ras2 (AbNova, Walnut, CA), followed by HRP-conjugated secondary Ab (Bio-Rad, Hercules, CA). To control for protein loading, blots were probed for GAPDH expression using mouse mAb 6C5 (American Research Products, Belmont, MA). Incubations with the primary Ab were overnight at 4°C with rocking, and incubations with secondary Ab were performed at room temperature for 45 min following thorough washing. Blots were visualized using Supersignal chemiluminescence substrate (Pierce) exposed to film (Kodak, Rochester, NY).
Macrophages were treated with 100 nM estradiol for 24 h prior to stimulation with LPS (10 ng/ml) for 1 h. Nuclear protein lysates were generated using the NE-PER kit (Pierce). Fifty micrograms of nuclear protein lysate were used, according to the manufacturer’s protocol, to analyze NF-κB p65 expression by ELISA (BioSource International, Camarillo, CA).
Culture supernatants were analyzed for TNF-α expression by ELISA (R&D Systems), according to the manufacturer's protocol. Samples stimulated with LPS were diluted 100-fold, and unstimulated samples were run undiluted.
RAW 264.7 cells were plated 24 h prior to transfection at a density of 2 × 105 cells per well in 24-well tissue-culture dishes. Cells were transfected with 100 nM premiRNA or negative control premiRNA (Ambion, Austin, TX) and 800 ng κB-Ras2 3′UTR luciferase plasmid or control plasmid (SwitchGear Genomics, Menlo Park, CA), using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection efficiency was normalized to Renilla luciferase activity by cotransfection of 100 ng pRL-TK expression vector (Promega). Cells were lysed 48 h posttransfection using passive lysis buffer (Promega). Luciferase activity was determined using the dual-luciferase reporter assay system (Promega) and the Berthold Centro LB960 luminometer. Data are the mean of at least three independent experiments and are represented as normalized relative light units.
Transient transfection of premiRNAs
U937 cells (obtained from American Type Culture Collection, Manassas, VA) were differentiated with PMA (6 ng/ml, Sigma-Aldrich) for 2 d. Cells were plated at a density of 8 × 105 cells per well in six-well culture dishes 24 h prior to transfection. Cells were transfected with 100 nM pre-microRNA (premiR)-125b, prelet-7a, or negative control premiRNA (Ambion), using FugeneHD transfection reagent (Roche, Indianapolis, IN). Forty-eight hours after transfection, total RNA was harvested and analyzed for κB-Ras2 expression. Data are representative of three independent transfections.
All experiments were performed using n = 3–4 individual donors, unless otherwise noted. Results are presented as mean ± SE. Statistical analysis was performed using a paired Student t test, and significance was achieved at p < 0.05.
Estradiol attenuates TNF-α production in human macrophages
Although previous reports showed that estradiol affects TNF-α production in murine macrophages (6, 7), it was unknown whether it influences TNF-α production in human macrophages. To determine the effect of estradiol on TNF-α production in human macrophages, cells were pretreated with estradiol at concentrations ranging from 0.001–100 nM for 24 h. Following estradiol treatment, cells were stimulated with LPS for an additional 12 h, and soluble TNF-α levels were measured in the culture supernatants. As demonstrated in Fig. 1A, estradiol pretreatment dose-dependently attenuated LPS-induced TNF-α production. TNF-α protein levels were undetectable in unstimulated cells in the presence and absence of estradiol (data not shown). Similarly, estradiol treatment decreased TNF-α mRNA levels in response to LPS (Fig. 1B). Data are presented as the percent of control to permit optimal appreciation of estradiol-mediated effects on TNF-α expression. Although the magnitude of the TNF-α response to LPS stimulation varied among donors, the percent by which estradiol pretreatment attenuated TNF-α production was consistent among individuals. This is in accordance with other reports of interindividual differences in LPS-induced TNF-α production (15–17).
To determine whether this effect was mediated by signaling through the ER, cells were pretreated with the ER antagonist ICI 182,780, alone or in combination with estradiol, prior to LPS stimulation. In accordance with our earlier observations, pretreatment with estradiol attenuated LPS-induced TNF-α expression. However, TNF-α levels were not inhibited in macrophages that had been pretreated with ICI 182,780 and estradiol (Fig. 1C). Incubation with the ER antagonist alone had no effect on TNF-α expression. These data demonstrate that estradiol attenuates LPS-induced TNF-α secretion by human macrophages in an ER-dependent manner.
Estradiol treatment attenuates LPS induced NF-κB signaling
NF-κB plays a central role in the regulation of inflammation and is a major downstream target of nuclear steroid receptors and signal transduction pathways. Stimulus-induced activation of NF-κB contributes to the enhanced production of many proinflammatory cytokines, including TNF-α. Macrophages treated with an NF-κB activation inhibitor produced significantly less TNF-α in response to LPS, confirming the importance of this signaling pathway in the TLR4/TNF-α signaling axis (Fig. 2A).
Given the importance of NF-κB in the regulation of inflammation and our observation that estradiol pretreatment attenuated LPS-induced TNF-α production, we hypothesized that estradiol modulates the activation of NF-κB in human macrophages. To test this hypothesis, we pretreated macrophages with estradiol prior to stimulation with LPS and assessed estradiol effects on phosphorylation of IκBα. As demonstrated in Fig. 2B, estradiol pretreatment inhibited LPS-induced IκBα phosphorylation, suggesting that estradiol reduces LPS-induced nuclear translocation of NF-κB. Importantly, estradiol treatment inhibited the degradation of total IκBα in response to LPS, resulting in enhanced expression of total IκBα following LPS stimulation. In the absence of stimulation, total and phospho-IκBα were unaltered by estradiol treatment, and the expression of IKKβ was similarly unaffected (Fig. 2B). Collectively, these data suggest that estradiol treatment attenuates NF-κB signaling through decreased IκBα phosphorylation and degradation.
To determine whether reduced IκBα phosphorylation and degradation results in decreased p65 nuclear translocation, macrophages were pretreated with or without estradiol and stimulated with LPS for 1 h. Nuclear lysates were prepared and analyzed for p65 expression by ELISA. As expected, nuclear p65 levels were low but detectable in unstimulated cells and were significantly increased with LPS treatment (Fig. 2C). Notably, pretreatment with estradiol markedly diminished LPS-induced nuclear p65 levels. These data demonstrate that estradiol attenuates NF-κB signaling through reduced IκBα phosphorylation and degradation, resulting in inhibited LPS-induced nuclear translocation of NF-κB.
Estradiol abrogates LPS-mediated inhibition of κB-Ras2
κB-Ras2 associates with IκBα and inhibits its degradation, resulting in decreased activation of NF-κB signaling (8, 9). Because estradiol inhibited NF-κB signaling at the levels of IκBα phosphorylation and degradation, we decided to test whether κB-Ras2 expression was modulated by estradiol in the context of LPS activation. Expression of κB-Ras2 has been demonstrated in a variety of tissues, including the heart, placenta, liver, and lung (8). However, it was unknown whether human macrophages express this protein and whether its expression is regulated by LPS. As demonstrated in Fig. 3A, κB-Ras2 mRNA expression decreases in response to LPS, reaching a nadir within 3–8 h of stimulation, and returns to baseline after 12 h of LPS treatment. Similarly, κB-Ras2 protein expression is maximally inhibited after 12 h of stimulation, and expression returns to unstimulated levels following 24 h of LPS treatment (Fig. 3B). Data shown are representative of four experiments. Although the magnitude of inhibition varied between 15 and 55%, in each instance tested LPS attenuated κB-Ras2 expression.
We next asked whether LPS-induced changes in κB-Ras2 expression were modulated by estradiol. Intriguingly, estradiol pretreatment abrogated the LPS-induced inhibition of κB-Ras2 mRNA in human macrophages (Fig. 3C). As shown in Fig. 3C and 3D, κB-Ras2 mRNA levels were slightly increased by estradiol in the absence of LPS, but protein levels were unaltered by estradiol in unstimulated cells. Immunoblot analysis was performed to determine how estradiol affects the expression of κB-Ras2 protein following activation with LPS. Fig. 3D demonstrates that estradiol-treated cells abrogated the LPS-induced decrease in κB-Ras2 expression and augmented κB-Ras2 expression compared with unstimulated cells after 12 and 24 h of LPS stimulation. These data demonstrate for the first time that estradiol modulates the LPS-regulated expression of κB-Ras2, a key negative regulator of NF-κB activation.
miR-125b and let-7a are coordinately regulated by LPS and estradiol
Because miRNAs have been identified as important regulators of NF-κB signaling (18–20), we next investigated whether κB-Ras2 is regulated by miRNAs. Using TargetScan analysis (10), we identified several putative miRNA-binding sites in the κB-Ras2 3′UTR. Let-7a and miR-125b were among the predicted interacting miRNAs. Let-7a directly targets the 3′UTRs of other RAS protein family members (21), and let-7e, another member of the let-7 family, is modulated by LPS in monocytes (22). miR-125b is also an LPS-regulated miRNA (23–25) and directly targets TNF-α mRNA in mice (25). However, there are no seed-binding regions in the 3′UTR of human TNF-α mRNA for miR-125b.
To determine whether let-7a and miR-125b regulate κB-Ras2, we first examined their expression patterns in response to estradiol and LPS by TaqMan real-time PCR. As depicted in Fig. 4A, let-7a expression was ∼17-fold greater than miR-125b in resting macrophages. We next performed a time course to determine how the expression of let-7a and miR-125b is altered by activation with LPS. The results of this study showed that let-7a was induced, and, conversely, miR-125b expression was decreased in response to LPS treatment (Fig. 4B, 4C). Although let-7a induction did not occur until after 12 h of LPS stimulation, miR-125b expression decreased more rapidly. These data suggest that the effects of these miRNAs occur sequentially and likely are not contemporaneous. To determine whether the regulation of let-7a and miR-125b is dependent on NF-κB activation, cells were treated with an NF-κB activation inhibitor for 1 h prior to stimulation with LPS. As demonstrated in Fig. 4D, the inhibition of NF-κB activation blocked the induction of let-7a by LPS, indicating that this pathway is necessary for let-7a expression. In contrast, miR-125b regulation by LPS occurs independent of NF-κB signaling, because the inhibition of NF-κB activation did not reverse the decrease in miR-125b expression (Fig. 4E). When macrophages were treated with estradiol prior to stimulation, the LPS-induced increase in let-7a expression was abrogated (Fig. 5A). Estradiol treatment also blocked the LPS-induced decrease in miR-125b expression (Fig. 5B). These data indicate that estradiol pretreatment reversed the effects of LPS on the expression of these miRNAs. Although estradiol had no effect on miR-125b levels in unstimulated cells, it significantly induced let-7a expression (Fig. 5A, 5B).
The predicted interactions of let-7a and miR-125b with the κB-Ras2 3′UTR are shown in Fig. 6A, and the secondary structure of miRNA/mRNA interactions, as predicted by the RNAhybrid program (26), are shown in Fig. 6B. To verify that miR-125b and let-7a directly target the κB-Ras2 3′UTR, we used a luciferase reporter system containing the 3′UTR of κB-Ras2. Transfection of let-7a decreased luciferase expression, indicating that let-7a suppresses κB-Ras2 3′UTR stability (Fig. 6C). Intriguingly, cells transfected with miR-125b expressed increased levels of luciferase in comparison with those transfected with control miRNA, demonstrating that miR-125b enhances the stability of the κB-Ras2 3′UTR (Fig. 6C). In agreement with this finding, macrophages transfected with prelet-7a had reduced levels of endogenous κB-Ras2, whereas the overexpression of premiR-125b led to enhanced κB-Ras2 expression (Fig. 6D). These findings indicate a coordinated regulation of let-7a and miR-125b by estradiol and LPS for the net effect of increasing κB-Ras2 expression in the presence of estradiol. We included a model depicting this mechanism of regulation in Fig. 7. Significantly, these data demonstrate estradiol regulation of miRNA in the context of inflammation in human macrophages.
This study demonstrates for the first time that estradiol attenuates LPS-induced TNF-α production in human macrophages through the modulation of NF-κB activation. We show that estradiol reduces NF-κB activation through the inhibition of IκBα phosphorylation and subsequent degradation, resulting in decreased p65 nuclear translocation. Although estradiol does not affect basal NF-κB signaling, the treatment of macrophages with estradiol prior to LPS activation led to an increase in the negative regulator of NF-κB signaling, κB-Ras2. Our data indicate that estradiol modulates the expression of two microRNAs (let-7a and miR-125b) that regulate the stability of the κB-Ras2 3′UTR. As depicted in Fig. 7A, in the absence of estradiol, LPS induces the expression of let-7a and inhibits the expression of miR-125b. This provides a net instability to the κB-Ras2 3′UTR. Estradiol reverses the effects of LPS on let-7a and miR-125b expression, for a net increase in κB-Ras2 stability (Fig. 7B). These data demonstrate a novel mechanism by which estradiol modulates NF-κB signaling and provide the first account of estradiol modulation of miRNA expression in human macrophages.
Numerous human and animal studies have shown that estradiol regulates proinflammatory cytokine production (12, 27–29). It is notable that males have a greater mortality due to sepsis versus premenopausal females; this can be attributed, at least in part, to elevated serum TNF-α levels (5). These observed differences in TNF-α expression implicate a role for sex hormones in the regulation of TNF-α expression. In this regard, plasma TNF-α values are inversely correlated with estradiol levels in female trauma patients (30). In addition, monocytes from premenopausal females who underwent oophorectomy produce elevated levels of TNF-α, concordant with a reduction in serum estradiol concentration. Treatment with exogenous estrogens abrogates this effect (31). Previous reports also showed that estradiol inhibits TNF-α in the murine macrophage cell line RAW 264.7 (7, 32) and in human THP-1 cells (33). In this study, we demonstrate that estradiol attenuates LPS-induced TNF-α in primary human monocyte-derived macrophages in a dose-dependent manner. These effects were mediated through the ER, as indicated by a lack of effect with receptor blockade.
As demonstrated in Fig. 1, estradiol modulates LPS-induced TNF-α expression in a dose-dependent manner. We chose to perform the remainder of our studies with 10−7 M estradiol because it demonstrated the maximal effect on TNF-α induction in macrophages. This concentration is physiologically relevant because it is consistent with levels present in the human female reproductive tract during ovulation. Estradiol levels in the ovarian vein, which drains directly into the human female reproductive tract, are 14–80-fold higher during ovulation than the levels measured in peripheral blood, and >100-fold higher than during the early proliferative phase of the menstrual cycle (34). Estrogen has been shown to accumulate in the cycling human uterine endometrium and vagina to ≥37 and 11 times that seen in plasma, respectively (35). Therefore, macrophages at these sites are routinely exposed to this concentration of estradiol. Intriguingly, 10−7 M estradiol also has physiologic relevance during pregnancy (36, 37); thus, our studies may have significant immunologic relevance at this time.
LPS binding to TLR4 activates the NF-κB signaling cascade, resulting in transcriptional upregulation of proinflammatory genes, including TNF-α. Fig. 2 demonstrates that estradiol treatment prior to LPS challenge results in a reduction in NF-κB signaling through the inhibition of IκBα phosphorylation and degradation. These findings are in agreement with the observed decrease in LPS-induced IKKβ activity by estradiol in endothelial cells (38), as well as the estradiol-mediated reduction of IκBα phosphorylation in cerebral ischemia (39). Decreased IκBα degradation impairs translocation of the NF-κB transcription factors to the nucleus. Indeed, our data show that estradiol pretreatment inhibits LPS-induced increases in nuclear p65 levels. These data are in agreement with observations by Ghisletti et al. (32) that estradiol also inhibits p65 nuclear translocation in RAW 264.7 cells. However, in contrast to our findings, these investigators found that estradiol reduced p65 translocation through the inhibition of nongenomic pI3K signaling, which was independent of IκBα degradation. The disparity in these results is most likely attributable to timing. The duration of estradiol treatment prior to LPS stimulation differed between their study, in which macrophages were treated with estradiol for 10 min prior to stimulation, and our study, in which cells were treated for 24 h prior to LPS treatment. The longer duration of estradiol treatment used in our study allows for transcriptional upregulation of genes that may be necessary for the effect of estradiol on IκBα, whereas estradiol pretreatment for 10 min is likely to mediate mainly nongenomic effects. Although estradiol was reported to inhibit NF-κB signaling in various cell types (40), the mechanisms by which this occurs are largely unknown. κB-Ras2 is a Ras-like protein that inhibits NF-κB signaling. Unlike the classic Ras proteins, κB-Ras2 lacks a C-terminal membrane attachment sequence and is constitutively bound to GTP. κB-Ras2 inhibits NF-κB signaling through direct binding to IκBα, decreasing its phosphorylation and consequent degradation (8). Our observation that estradiol inhibited NF-κB signaling at the level of IκBα prompted us to examine how κB-Ras2 expression is modulated by estradiol and LPS. Although relatively little is known about the regulation of κB-Ras2, it was recently identified as a heat shock-responsive gene in THP-1 cells (41), and it was shown to be regulated transcriptionally by FOXO3a in HUVECs (42). Fig. 3 demonstrates that κB-Ras2 is expressed in human macrophages and is downregulated by LPS at the mRNA and protein levels. Given these findings, we propose that κB-Ras2 may serve as a basal inhibitor of NF-κB signaling in the absence of TLR stimulation. In this study, we demonstrated that estradiol pretreatment abrogates the LPS-induced decrease in κB-Ras2 expression, as well as augments the expression of κB-Ras2 compared with unstimulated cells. We believe that estradiol-mediated upregulation of LPS-modulated κB-Ras2 represents a novel mechanism for the inhibition of NF-κB signaling in human macrophages.
Activation of macrophages with LPS leads to changes in the expression of miRNAs that regulate molecules involved in TLR signaling. Taganov et al. (20) described negative feedback on NF-κB signaling through the suppression of IL-1R–associated kinase-1 and TNFR-associated factor-6, which is mediated by miR-146a. In addition, miR-199a targets IKKβ in epithelial ovarian cancer cells (18), and IκB ζ expression is suppressed by miR-124a in Hep2G cells (19). Notably, miR-9 suppresses NFΚB1 expression in monocytes and neutrophils and is induced in response to LPS (22). To determine the role of miRNAs in the regulation of κB-Ras2 by estradiol and LPS, we used TargetScan analysis of the 3′UTR of κB-Ras2 mRNA and identified let-7a and miR-125b as potentially interacting miRNAs. Intriguingly, the 3′UTRs of the Ras family members HRAS, KRAS, and NRAS are also direct targets of let-7a (21). Fig. 4 illustrates the reciprocal regulation of let-7a and miR-125b in response to LPS stimulation in human macrophages. Our observation that miR-125b expression is decreased by LPS in human cells corroborates the recent finding that miR-125b is downregulated in activated mouse macrophages (24). The changes in let-7a and miR-125b expression occur with different kinetics, suggesting that the effects of these miRNAs on κB-Ras2 are not additive but sequential. Because κB-Ras2 seems to serve as a basal inhibitor of TLR signaling that prevents NF-κB activation in the absence of stimulation, the regulation of miR-125b early after LPS stimulation and let-7a later, allows for multiple phases of κB-Ras2 regulation. Our data demonstrate that although let-7a induction is dependent on NF-κB signaling, miR-125b regulation in response to LPS occurs independently of NF-κB activation (Fig. 4D, 4E). In addition to the NF-κB pathway, TLR ligation leads to the activation of several other signaling pathways, including MAPK and pI3K. Therefore, one or more of these mechanisms likely regulates the miR-125b response to LPS. In support of this, Androulidaki et al. (24) recently showed that Akt signaling is necessary for the LPS-mediated decrease in miR-125b expression in murine peritoneal and bone marrow-derived macrophages.
Although recent work implicates a role for estradiol in the regulation of miRNAs (43), its effect on miRNA expression in the context of inflammation is largely unknown. In this study, we demonstrate that estradiol pretreatment of human macrophages inhibits the LPS-mediated induction of let-7a and the inhibition of miR-125b expression. Given our observation that κB-Ras2 expression is also decreased by LPS stimulation, we hypothesized that let-7a and miR-125b have opposing effects on the stability of the κB-Ras2 3′UTR. We confirmed this hypothesis using the κB-Ras2 3′UTR luciferase reporter system and showed that let-7a overexpression suppressed the κB-Ras2 3′UTR, whereas miR-125b enhanced its stability. miRNAs modulate posttranscriptional gene expression by inducing changes in the rates of mRNA decay and/or translation of target genes. Our results demonstrate that let-7a and miR-125b regulate mRNA expression of κB-Ras2 (Fig. 6D). These findings suggest that these miRNAs alter κB-Ras2 expression through the modulation of mRNA stability; however, it is also possible that these miRNAs regulate κB-Ras2 expression through changes in rates of translation. Although miRNAs are mainly thought to suppress gene expression through mRNA degradation or translational repression, recent studies have shown that miRNAs can also stabilize their target messages (25, 44, 45).
miR-125b dysregulation is observed in various types of cancer, including acute myeloid leukemia (46) and breast cancer (47). In addition, let-7a is a bona fide tumor-suppressor miRNA and is dysregulated in cancers of the lung and breast (21, 48) and in primary effusion lymphoma (49). Intriguingly, miR-125b-1 and let-7a-1 localize to the 11q24.1 locus (www.atlasgeneticsoncology.org). Given that 11q24.1 is a susceptibility locus for chronic lymphocytic leukemia (50) and that deletions in the 11q23–11q24 region are common in cervical, breast, and ovarian cancer (51–55), it is tempting to speculate that dysregulation of miR-125b and let-7a together contribute to the pathogenesis of disease.
We report, for the first time, regulation of miRNAs by estradiol in LPS-activated human macrophages. We show that LPS downregulation of κB-Ras2 expression occurs in association with reciprocal regulation of let-7a and miR-125b. These studies implicate estradiol-mediated regulation of LPS-modulated κB-Ras2 expression as a novel mechanism for NF-κB inhibition and suggest that estradiol regulation of miRNAs serves as an important factor in controlling inflammation.
We thank Allan Munck, Mark Yeager, and Jane Collins for helpful discussions and critical review of the manuscript.
Disclosures The authors have no financial conflicts of interest.
This work was supported by National Institutes of Health Grant RO1AI051547 (to P.M.G.) and the Centers of Biomedical Research Excellence P20 RR 016437 (to P.M.G. and P.A.P.). P.A.P. received Prouty Pilot Project funds provided by the Norris Cotton Cancer Center at Dartmouth-Hitchcock Medical Center. A.J.M. received support from a National Institutes of Health Autoimmunity and Connective Tissue Training Grant (T32AR007576).