Abstract
Ethanol-mediated injury, combined with gut-derived lipopolysaccharide (LPS), provokes generation of proinflammatory cytokines in Kupffer cells, causing hepatic inflammation. Among the mediators of these effects, miR-217 aggravates ethanol-induced steatosis in hepatocytes. However, the role of miR-217 in ethanol-induced liver inflammation process is unknown. Here, we examined the role of miR-217 in the responses to ethanol, LPS, or a combination of ethanol and LPS in RAW 264.7 macrophages and in primary Kupffer cells. In macrophages, ethanol substantially exacerbated LPS-mediated induction of miR-217 and production of proinflammatory cytokines compared with LPS or ethanol alone. Consistently, ethanol administration to mice led to increases in miR-217 abundance and increased production of inflammatory cytokines in isolated primary Kupffer cells exposed to the combination of ethanol and LPS. miR-217 promoted combined ethanol and LPS-mediated inhibition of sirtuin 1 expression and activity in macrophages. Moreover, miR-217–mediated sirtuin 1 inhibition was accompanied by increased activities of two vital inflammatory regulators, NF-κB and the nuclear factor of activated T cells c4. Finally, adenovirus-mediated overexpression of miR-217 led to steatosis and inflammation in mice. These findings suggest that miR-217 is a pivotal regulator involved in ethanol-induced hepatic inflammation. Strategies to inhibit hepatic miR-217 could be a viable approach in attenuating alcoholic hepatitis.
Alcoholic liver disease (ALD) is associated with the spectrum of pathology that ranges from excess accumulation of fat within the liver (steatosis), inflammation (hepatitis), fibrosis/cirrhosis, liver failure, and hepatocellular cancer.1 Activation of Kupffer cells (KCs), the resident macrophages, by elevated gut-derived lipopolysaccharide (LPS) plays a central role in the development of alcoholic hepatitis.1–3 LPS is a known potent inflammatory inducer in both rodent models of ALD and human alcoholics.2,3 LPS enters the liver via the portal vein and activates KCs, which stimulates generation of proinflammatory cytokines. Ethanol also sensitizes KCs in response to LPS, enhancing LPS-stimulated production of inflammatory cytokines, which exacerbates liver injury. Therefore, ethanol and LPS co-exist and contribute, additively or synergistically, to the pathogenesis of alcoholic hepatitis.
Sirtuin 1 (SIRT1) is an NAD+-dependent class III protein deacetylase that exerts anti-inflammatory effects via coordinated regulation of several signaling molecules such as NF-κB and the nuclear factor of activated T cells c4 (NFATc4), two pivotal transcription factors involved in regulation of proinflammatory cytokines.4–6 In recent years, SIRT1 has emerged as an important player in alcohol-induced development of steatohepatitis in the liver.6–9 The inhibition of SIRT1 by three major putative inducers of ALD, namely LPS, acetaldehyde, and acetate, results in a substantial increase in the generation of proinflammatory cytokines via disruption of SIRT1-mediated deacetylation of NF-κB in macrophages.9 Nevertheless, the mechanisms underlying the interactions between ethanol and LPS leading to aberrant SIRT1 signaling and subsequent hepatic inflammation remain incompletely understood.
miRNAs are a class of small noncoding RNAs that induce gene silencing by either inhibiting the expression of target mRNAs or by causing mRNA degradation.10,11 A broad array of aberrant hepatic miRNAs such as miR-34a, miR-214, miR-155, miR-212, and miR-199 are associated with the pathogenesis of ALD in rodent and human studies.11 We identified miR-217, a known endogenous inhibitor of SIRT1, as a specific target of ethanol action in the liver.12,13 We demonstrated that miR-217 abundance was drastically and specifically increased in both hepatocytes exposed to ethanol and chronically ethanol-fed mice.12 More importantly, we discovered that ethanol-mediated induction of miR-217 results in SIRT1 suppression and subsequently promotes development of steatosis in hepatocytes and in mice.12 However, the role of aberrant miR-217 in macrophages or KCs and its possible regulation by alcohol is unknown. The possibility that ethanol mediates pleiotropic effects through a range of cell types is important in view of the evidence demonstrating that miR-217 deregulation is associated with a range of conditions, including aging, diabetes, Tat-induced HIV-1 transactivation, and recurrence of liver cancer.13–17
Here, we investigated cell-specific mechanisms and pathways that accompany altered expression of miR-217 in response to ethanol and LPS in cultured macrophages and primary KCs.
Materials and Methods
Reagents, Antibodies, and Plasmids
LPS (tissue culture-tested, L-2654) and ethanol were purchased from Sigma Chemical (St. Louis, MO). SIRT1, phosphorylated AMP-activated kinase α (AMPKα), AMPKα, NF-κBp65, acetylated histone H3-Lys 9 (Ac-histone H3-Lys9), and histone H3, Ac-histone H4-Lys5, and Ac-histone H4-Lys8 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Acetyl-NF-κBp65 antibody was from Abcam (Cambridge, UK). NFATc4 and β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). NF-κB-responsive reporter-3xκB luciferase plasmid and wild-type SIRT1 (SIRT1wt) expression plasmids were kind gifts from Dr. Marty W. Mayo (University of Virginia, Charlottesville, VA). The small silencing SIRT1 plasmid (SIRT1shRNA) was purchased from Santa Cruz Biotechnology, Inc. The luciferase vector, including 3′ untranslated region (UTR) of human SIRT1 containing the SIRT1–miR-217 response elements (SIRT1-3′ UTR) was purchased from Addgene Inc. (Cambridge, MA). pcDNA 6.2-GW/miR-217 and pcDNA 6.2-GW/scmiR-217 expression plasmids were kind gifts from Dr. Gregory M. Vercellotti (University of Minnesota, Minneapolis, MN). miRIDIAN miR-217 mimic (miR-217) and miRIDIAN hairpin inhibitor miR-217 (anti-miR-217) were purchased from Thermo Scientific (Marietta, OH). Vectors expressing adenovirus (Ad)-green fluorescent protein (GFP), Ad-miR-217, Ad–anti-miR-217, and Ad-SIRT1wt were obtained from Vector BioLabs, Inc. (Malvern, PA). Ad–lipin-1α was a kind gift from Dr. Brian Finck (Washington University School of Medicine, St. Louis, MO).
Cell Culture
The murine RAW264.7 macrophages were obtained from ATCC (Manassas, VA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 63 μg/mL penicillin G. For treatments, cells were plated onto six-well plates. At 80% to 90% confluence, the cells were incubated for 16 hours in serum-free RPMI 1640 medium.9
ELISA Analysis
Tumor necrosis factor (TNF)-α or IL-6 concentration in culture media of RAW 264.7 macrophages or primary KCs was measured with a mouse ELISA kit (eBioscience, San Diego, CA) according to the manufacturer's protocol. Analysis was performed with a Synergy HT microplate reader (BioTek, Winooski, VT).
Quantitative Real-Time RT-PCR Analysis
Total RNA isolation was from cells or liver samples. Quantitative real-time RT-PCR amplification or miRNA analysis was performed as described previously.6–9 The primer sets were either purchased from SuperArray Bioscience (Frederick, MD) or synthesized as previously described.9,12 The relative amount of target mRNA was calculated by the comparative threshold cycle method by normalizing target mRNA threshold cycle to those for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). U6 small nuclear RNA was used as an internal control for miRNA analysis.
SIRT1 Deacetylase Activity
SIRT1 activity was measured by a SIRT1 fluorometric kit (AK-555; Biomol, Plymouth Meeting, PA) as described previously.9,12
Immunoblot Analysis
Western blot analyses were performed with cell extract separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose filters. The protein bands were quantified on a PhosphorImager and ImageQuant (Amersham Biosciences, Piscataway, NJ) software analysis.
Luciferase Assays, Transfections, and Ad Infection
Cell transfection assays were performed as described.9 Briefly, plasmids were transiently transfected into cells by Lipofectamine reagent (Invitrogen, Carlsbad, CA). Luciferase assays were performed with cell extracts, and the results were averaged to represent a single data point for each transfection. β-Galactosidase was used as an internal control to correct for transfection efficiency. The replication-deficient Ads were amplified in human embryonic kidney 293 cells and purified by Adenopure kits. Cells were infected with Ads for 36 to 48 hours before the experiments.18,19
ChIP Assay
The chromatin immunoprecipitation (ChIP) assay was performed with the ChIP assay kit from Upstate Biotechnology (Lake Placid, NY) as described.20 Briefly, RAW 264.7 macrophages were cross-linked, and anti-NFATc4 or anti–Ac-histone H3-Lys9 antibody was added to the sonicated chromatin solution and incubated. After reversal of the cross-linking, the DNA was purified. The samples were subjected to quantitative real-time RT-PCR by using the following primers that amplify a region that contained NFATc4 binding sites of TNF-α (forward, 5′-CGATGGAGAAGAAACCGAGACAGA-3′; reverse, 5′-AGGGAGCTTCTGCTGGCTGGCTGT-3′).21 The primer set (forward, 5′-GGCTCACAACCATCTATAATCAGGT-3′; reverse: 5′-ACAGCTTTCTGCTTGCATGTATG-3′) for the GAPDH was used as a control. The results were normalized to control IgG, GAPDH, and input DNA.
Quantification of Cellular ROS
Levels of intracellular reactive oxygen species (ROS) were measured with 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate by using ROS detection reagents (Invitrogen). Briefly, cells were incubated with 10 μmol/L 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate in phosphate-buffered saline at 37°C for 30 minutes. Cells were then placed in fresh Phenol Red-free culture medium. The cells' fluorescence was measured with a Synergy HT microplate reader (BioTek).
Animal Studies and Isolation of Primary KCs
The detailed Gao-binge (Chronic+binge) ethanol feeding protocol has been described.22 Ten- to 12-week-old male C57BL/6J mice were divided into two dietary groups: pair-fed control diet and ethanol-containing diet. Ethanol groups were fed a liquid diet that contained 5% ethanol for 10 days, whereas the control mice were pair-fed to their ethanol-fed counterparts for 10 days. At day 11, mice in the ethanol groups were gavaged a single dose of ethanol (5 g/kg body weight, 20% ethanol), whereas mice in the control groups were gavaged an isocaloric dose of dextrin maltose. The mice received anesthesia with 100 mg/kg ketamine, and KCs were isolated as described.23 Livers were perfused with saline solution for 10 minutes, followed by in vivo digestion with liberase enzyme for 5 minutes and in vitro digestion for 30 minutes. The non-hepatocyte content was separated by Percoll gradient and centrifuged. The intercushion fraction was adhered to plastic in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. The nonadherent fraction was washed, and the adherent KC population was adjusted to 2 × 106/mL in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Depending on experimental conditions, KCs from three to six mice were pooled for each experiment. For in vitro stimulation, KCs were stimulated with 50 mmol/L ethanol or 100 ng/mL LPS or both for 6 hours.
In Vivo Ad-Mediated Gene Transfer
During the 10 days Gao-binge (Chronic+binge) ethanol treatment period, overexpression of miR-217 or anti-miR-217 in the mouse livers was accomplished via tail vein injection of Ad-GFP (control), Ad–miR-217, or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) to male C57BL/6J mice (10- to 12-week-old) twice on day 1 and day 5.24 On the final day mice were sacrificed, and tissues were rapidly taken and freshly frozen in liquid nitrogen and stored at −80°C. Parts of tissues were fixed for histology and immunohistochemistry. The local institutional animal care and use committee approved all in vivo animal protocols.
Statistical Analysis
Data are expressed as means ± SEM. Multiple comparisons were evaluated by analysis of variance followed by Tukey's multiple-comparison procedure with P < 0.05 being significant.
Results
Ethanol Exacerbates LPS-Mediated Up-Regulation of miR-217 in RAW 264.7 Macrophages and Primary KCs
We found that ethanol at 50 mmol/L and LPS at 100 ng/mL generated optimal and reproducible effects in both murine RAW 264.7 macrophages and primary KCs, and these concentrations were used in subsequent experiments.9,12 We then investigated miR-217 expression in RAW 264.7 macrophages in response to ethanol, LPS, or a combination of ethanol and LPS (E+L) exposure. Treatment with ethanol or LPS alone resulted in a significant increase in miR-217 abundance compared with controls in macrophages. Moreover, ethanol exacerbated LPS-mediated induction of miR-217 (approximately 80-fold) compared with controls (Figure 1A). The abundance of both miR-33 and miR-34a was also increased, albeit to a lesser degree than miR-217, in response to ethanol, LPS, or E+L (Figure 1A).
Figure 1.
Up-regulation of miR-217 by ethanol or LPS in RAW 264.7 macrophages and mouse primary KCs. A: RAW264.1 cells were treated with 50 mmol/L ethanol, 100 ng/mL LPS, or E+L for 18 hours, and miRNA-217 levels were measured with RT-qPCR. B: Primary KCs isolated from pair-fed or ethanol-fed mice were stimulated with ethanol, LPS, or E+L for 6 hours, and miR-217 levels were measured. C: Relative mRNA levels of IL-6 in KCs isolated from livers of mice fed with or without ethanol and stimulated with ethanol, LPS, or E+L. D: Relative mRNA levels of TNF-α in KCs isolated from livers of mice fed with or without ethanol stimulated with ethanol, LPS, or E+L. Data are expressed as means ± SEM. n = 3 to 5experiments. Means without a common letter differ, P < 0.05. EtOH, ethanol; E+L, 50 mmol/L ethanol plus 100 ng/mL LPS; KC, Kupffer cell; LPS, lipopolysaccharide; RT-qPCR, quantitative real-time RT-PCR; TNF, tumor necrosis factor.
We next assessed whether the effects of ethanol observed in macrophages also occurred in primary KCs from livers of mice after a chronic-binge ethanol feeding protocol.22 Primary KCs isolated from livers of pair-fed control mice showed significantly increased miR-217 levels in response to ethanol or LPS compared with controls without in vitro stimulation (Figure 1B). As expected, ethanol exposure significantly exacerbated LPS-mediated induction of miR-217 compared with controls (Figure 1B).
Although miR-217 levels were significantly higher in KCs of mice fed with ethanol compared with mice fed with the control diet, ethanol, LPS, or E+L stimulation significantly amplified miR-217 expression in KCs isolated from ethanol-fed mice compared with pair-fed control mice (Figure 1B). Taken together, these data demonstrate that ethanol exacerbates LPS-mediated up-regulation of miR-217 in both RAW 264.7 and primary KCs.
Ethanol Promotes Production of a Panel of Proinflammatory Cytokines in RAW 264.7 Macrophages or Primary KCs Exposed to LPS
Consistent with the changes noted in miR-217 abundance, the mRNA abundance of target genes, TNF-α and IL-6, were in turn significantly increased in primary KCs stimulated with ethanol, LPS, or E+L compared with controls (Figure 1, C and D). Although the primary KCs isolated from ethanol-fed mice displayed significantly increased mRNA levels of TNF-α or IL-6 compared with pair-fed controls, LPS, ethanol, or E+L stimulation in KCs isolated from ethanol-fed mice resulted in significantly higher TNF-α or IL-6 at mRNA levels than in KCs from LPS-, ethanol-, or E+L-stimulated pair-fed control mice (Figure 1, C and D). In vitro ethanol stimulation in KCs isolated from ethanol-fed mice further augmented LPS-stimulated IL-6 (Figure 1C). There were also trended increases in LPS-stimulated TNF-α by in vitro ethanol stimulation in KCs; however, the changes did not reach statistical significance (Figure 1D).
In RAW 264.7 macrophages, miR-217 overexpression or E+L treatment significantly increased mRNA expression of several inflammatory cytokines [IL-1β, interferon-γ, monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase] compared with controls (Supplemental Figure S1). Moreover, forced overexpression of miR-217 augmented the E+L-mediated increases in mRNA expression of IL-1β, MCP-1, and inducible nitric oxide synthase but interferon-γ (Supplemental Figure S1). Further, overexpression of Sc–miR-217 did not alter mRNA expression of these cytokines (Supplemental Figure S1).
Collectively, these results suggest that miR-217 modulates expression of a panel of inflammatory cytokines in macrophages exposed to ethanol, LPS, or E+L.
miR-217 Exacerbates Impairment of SIRT1 Induced by E+L in RAW 264.7 Macrophages
Overexpression of miR-217 or E+L treatment in RAW 264.7 cells significantly inhibited SIRT1-3′ UTR reporter activity, SIRT1 expression (mRNA and protein) levels, and deacetylase activities of SIRT1 compared with Sc–miR-217 transfected controls (Figure 2 and Supplemental Figure S2A). Again, miR-217 exacerbated the E+L-mediated impairment of SIRT1 (Figure 2 and Supplemental Figure S2A).
Figure 2.
miR-217 exacerbates impairment of SIRT1 induced by a combination of E+L in RAW 264.7 macrophages. A: RAW 264.7 macrophages were transfected with a SIRT1-3′ UTR reporter and expression plasmids of control vector, miR-217, Sc-miR-217, and β-galactosidase (internal control). E+L was added for 18 hours. Forty-eight hours after transfection, cells were harvested, and luciferase and β-galactosidase activities were determined. B: RAW 264.7 cells were transfected with plasmids of control vector, miR-217, or Sc-miR-217. E+L was then added for 18 hours. RT-qPCR was used to estimate relative mRNA levels of SIRT1. C: Cell extracts of RAW 264.7 cells transfected with plasmids of control vector, miR-217, or Sc-miR-217 with or without E+L were immunoblotted with a SIRT1, p-AMPKα, AMPKα, or β-actin antibody. D: SIRT1 deacetylase activity was measured in RAW 264.7 cells transfected with plasmids of control vector, miR-217, or Sc-miR-217 with or without E+L. SIRT1 deacetylase activity is represented as AFUs. Data are expressed as means ± SEM. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. AFU, arbitrary fluorescence unit; AMPKα, AMP-activated kinase α; E+L, ethanol and lipopolysaccharide; p-AMPKα, phosphorylated-AMPKα; RT-qPCR, quantitative real-time RT-PCR; SIRT1, sirtuin 1; UTR, untranslated region.
SIRT1 regulates AMPK, another important anti-inflammatory regulator.5 miR-217 or E+L inhibited AMPK activity as demonstrated by a reduced phosphorylated AMPKα/AMPKα ratio, and E+L-mediated AMPK inhibition was exacerbated by miR-217 overexpression compared with Sc–miR-217 controls (Figure 2C and Supplemental Figure S2B).
By contrast, SIRT1 and AMPK expression were at least partially restored by miR-217 antisense-mediated oligonucleotide inhibition (anti-miR-217) in RAW 264.7 cells exposed to E+L (Figure 3A and Supplemental Figure S2, C and D). Inhibition of miR-217 expression in turn reduced generation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, and interferon-γ (Figure 3, B and C). Note that E+L-mediated inducible nitric oxide synthase induction was not affected by anti–miR-217, implying that miR-217–independent mechanisms may be involved (Figure 3B). Note that the efficacy of Ad–miR-217 or Ad–anti-miR-217 in macrophages was excellent (Supplemental Figure S2E).
Figure 3.
Inhibition of miR-217 by anti-miR-217 alleviates impairment of SIRT1 signaling induced by E+L in RAW 264.7 cells. A: RAW 264.7 macrophages were transfected with control vector or anti-miR-217. Forty-eight hours after transfection, E+L was added. After incubation for 24 hours, cell extracts were immunoblotted with a SIRT1, p-AMPKα, AMPKα, or β-actin antibody. B: Relative mRNA levels of inflammatory cytokines, including IL-1β, MCP-1, and IFN-γ, in RAW 264.7 cells transfected with plasmids of control or anti-miR-217 treated with or without E+L. C: Medium levels of TNF-α in RAW 264.7 cells transfected with plasmids of control or anti-miR-217 treated with or without E+L. Data are expressed as means ± SEM. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. AMPKα, AMP-activated kinase α; E+L, ethanol plus lipopolysaccharide; IFN, interferon; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemotactic protein 1; p-AMPKα, phosphorylated-AMPKα; SIRT1, sirtuin 1; TNF, tumor necrosis factor.
Taken together, these results again support a role for miR-217 in E+L-mediated production of inflammatory cytokines via SIRT1 inhibition in macrophages.
miR-217 Promotes Activation of NF-κB and NFATc4 in Macrophages Stimulated by E+L
Previous work demonstrated that SIRT1 regulates the expression of both NF-κB and NFATc4 in the induction of inflammatory cytokine expression.5,6,25 Consistent with these findings, miR-217 overexpression or E+L treatment was associated with increased acetylation of NF-κB and enhanced NF-κB transcriptional activity (Figure 4A and Supplemental Figure S3A). Moreover, miR-217 overexpression or E+L treatment led to nuclear accumulation of NFATc4 and decreased the abundance of cytoplasmic NFATc4 (Figure 4B and Supplemental Figure S3B). In particular, miR-217 overexpression along with E+L exacerbated activation of both NF-κB and NATc4 (Supplemental Figure S3, A and B).
Figure 4.
miR-217 promotes activation of both nuclear NF-κB and NFATc4 in macrophages exposed to E+L. A: Representative Western blot analyses of Ac-NFκB in RAW 264.7 cells transfected with plasmids of control vector, miR-217 treated with or without E+L. B: Representative Western blot analyses of cytosol or nuclear NFATc4 protein levels in RAW 264.7 cells transfected with plasmids of control vector, miR-217 treated with or without E+L. C: ChIP assays were performed in RAW 264.7 cells infected with Ad-GFP, Ad-SIRT1wt, Ad-lipin 1α, or treated with 5 μg/mL CsA in the presence or absence of E+L for 18 hours. Chromatin was immunoprecipitated with preimmune rabbit IgG, anti–acetyl-histone H3-Lys9, anti-NFATc4 antibody. Immunoprecipitates were subjected to PCR with a primer pair specific to the TNF-α promoter containing NFATc4 binding site. D: ChIP assays were performed in RAW 264.7 cells infected with Ad-GFP, Ad-miR-217 along with Ad-SIRT1wt, Ad-lipin-1α, or treated with 5 μg/mL CsA. Chromatin was immunoprecipitated with preimmune rabbit IgG, anti–acetyl-histone H3-Lys9, anti-NFATc4 antibody. Immunoprecipitates were subjected to PCR with a primer pair specific to the TNF-α promoter containing NFATc4 binding site. Data are expressed as means ± SEM. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. Ac, acetylated; Ad, adenovirus; ChIP, chromatin immunoprecipitation; CsA, cyclosporine; E+L, ethanol plus lipopolysaccharide; GFP, green fluorescent protein; NFATc4, nuclear factor of activated T cells c4; SIRT1wt, sirtuin 1 wild-type; TNF, tumor necrosis factor.
Consistent with the hypothesis that SIRT1 is a key mediator of these effects, we found that miR-217 overexpression in macrophages caused hyperacetylation of histone H3-Lys9 and histone H4-Lys5 (data not shown).
Involvement of SIRT1–Lipin-1α Axis in Increased Association of Ac-Histone H3-Lys9 and NFATc4 with TNF-α Promoter in Macrophages Regulated by miR-217 or E+L
The SIRT1–lipin-1α axis is known to be regulated by miR-217.12 The effect of miR-217 or E+L treatment on the binding of Ac-histone H3-Lys9 or NFATc4 to a known NFATc4 target gene, TNF-α, was determined in RAW 264.7 cells. ChIP assays with Ac-histone H3-Lys9 and NFATc4 antibodies were performed on RAW 264.7 cells infected with Ad-GFP, Ad–miR-217, Ad-SIRT1wt, Ad–lipin-1α in the presence or absence of E+L, respectively. Immunoprecipitated DNA was then amplified by quantitative real-time PCR with primers for the TNF-α promoter region containing NFATc4 response elements. Although immunoprecipitation of either Ac-histone H3 or NFATc4 enriched recovery of genomic DNA from the promoters of TNF-α (but not GAPDH controls in macrophages), E+L or miR-217 significantly increased association of Ac-histone H3-Lys9 or NFATc4 with TNF-α promoter compared with the controls (Figure 4, C and D). As expected, 5 μg/mL cyclosporine, a known NFATc4 inhibitor,21 partially blocked the increase in association of Ac-histone H3-Lys9 or NFATc4 with TNF-α promoter (Figure 4, C and D).
Co-expression of SIRT1wt or lipin-1α largely abolished E+L- or miR-217–mediated increases in the yield of Ac-histone H3-Lys9 or NFATc4 found at the promoter of TNF-α (Figure 4, A and B). As a net result, the synergistically enhanced serum TNF-α or IL-6 protein levels observed after miR-217 plus E+L treatment were abrogated by co-expression of SIRT1wt or lipin-1α (Supplemental Figure S3, C and D).
Taken together, these findings suggest a cascade of regulatory effects in ethanol-mediated inflammation that includes a central role for the miR-217–SIRT1 axis.
miR-217 Promotes Generation of TGF-β or ROS Stimulated by E+L in Macrophages
We next explored some potential mechanisms whereby miR-217 itself may be modulated in ethanol-mediated injury, particularly because miR-217 is known to be up-regulated by transforming growth factor (TGF)-β.17,26 Indeed, addition of 10 ng/μL TGF-β to macrophages substantially increased miR-217 expression up to 14-fold compared with controls (Figure 5A). Moreover, hydrogen peroxide also dose dependently induced miR-217 expression in macrophages (Figure 5B). In addition, overexpression of miR-217 in macrophages significantly induced TGF-β mRNA and ROS production; E+L treatment robustly increased TGF-β or ROS by approximately threefold (Figure 5, C and D). More importantly, miR-217 overexpression significantly augmented the E+L-mediated increases of TGF-β or ROS in macrophages (Figure 5, C and D). These findings suggest that there may be feed-forward regulation of miR-217 through mechanisms that involve TGF-β and/or ROS.
Figure 5.
miR-217 promotes TGF-β or ROS production in macrophages exposed to E+L. A: Relative miR-217 levels in RAW 264.7 macrophages treated with indicated concentrations of TGF-β for 18 hours. B: Relative miR-217 levels in RAW 264.7 macrophages treated with indicated concentrations of hydrogen peroxide for 18 hours. C: Relative TGF-β mRNA levels in RAW 264.7 macrophages transfected with control vector, miR-217, or Sc-miR-217 in the presence or absence of E+L. D: ROS levels in RAW 264.7 macrophages transfected with control vector, miR-217, or Sc-miR-217 in the presence or absence of E+L. Data are expressed as means ± SD. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. DCF, dichlorodihydrofluorescein; E+L, ethanol plus lipopolysaccharide; ROS, reactive oxygen species; TGF, transforming growth factor.
Ad-Mediated Elevation of miR-217 Causes Steatosis and Provokes Inflammation in the Livers of Mice
To determine more directly a possible in vivo role of hepatic miR-217, we used Ad–miR-217), miR-217 inhibitor (Ad–anti-miR-217), or Ad-GFP controls through intravenous injection.24 As expected, miR-217 abundance was significantly reduced in the mice injected with Ad–anti-miR-217, whereas miR-217 abundance was increased in the livers of mice injected with Ad-miR-217 (Supplemental Figure S4A).
Histologic analysis of the liver sections verified accumulation of lipid droplets in the livers of mice injected with Ad–miR-217 compared with the livers of Ad-GFP control or controls injected with Ad–anti-miR-217 (Figure 6A). Moreover, mice expressing miR-217 displayed significantly increased levels of hepatic triglyceride and cholesterol and elevated serum alanine aminotransferase compared with Ad-GFP or mice injected with Ad–anti-miR-217 (Figure 6, B and C, and Supplemental Figure S4, A and B).
Figure 6.
Ad-mediated elevation of miR-217 in the liver causes steatosis and provokes inflammation in mice. A: Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) was administered to 10- to 12-week-old male C57BL/6J mice twice on day 1 and day 5. After a 10-day period, mice were sacrificed. Immunohistochemical staining of the liver sections for H&E or F4/80+ was used. B: Hepatic TG levels. C: Hepatic cholesterol levels. D: Relative hepatic mRNA levels of TNF-α, Mip-1α, MCP-1, COX-2, and IFN-γ. E: Relative hepatic mRNA levels of TGF-β. Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; COX, cyclooxygenase; GFP, green fluorescent protein; H&E, hematoxylin and eosin; IFN, interferon; IHC, immunohistochemistry; MCP, monocyte chemotactic protein; Mip, macrophage inflammatory protein; TG, triglyceride; TGF, transforming growth factor; TNF, tumor necrosis factor.
Although no increases in hepatic F4/80+ staining, a marker for activated macrophages including KCs, were seen in mice that received Ad–anti-miR-217 or Ad-GFP controls, increased abundance was noted by immunohistochemical staining of F4/80+ in the livers of mice injected with Ad–miR-217, findings consistent with the suggestion that miR-217 overexpression provokes hepatic inflammation (Figure 6A).
The utility of the Ad–miR-217 vector in exacerbating liver injury was further verified by analyzing the mRNA expression of a panel of proinflammation cytokines. The mRNA abundance of macrophage inflammatory protein-1α, cyclooxygenase 2, interferon-γ, MCP-1, TGF-β, and circulating TNF-α levels were all significantly elevated in mice treated with Ad–miR-217 compared with controls treated with Ad–anti-miR-217 or Ad-GFP (Figure 6, D and E, and Supplemental Figure S4C).
In addition, mRNA expression of hepatic serum amyloid A1 and lipocalin 2 were elevated nearly 50- and 20-fold, respectively, in the livers of mice injected with Ad–miR-217 compared with Ad–anti-miR-217 or Ad-GFP controls (Supplemental Figure S5, A and B). Collectively, these results reinforce our conclusions that imply a causal role of miR-217 in the development of liver steatosis and inflammation in mice.
We further performed additional experiments to examine the effect of Ad–miR-217 or Ad–anti-miR-217 in the setting of ethanol feeding in mice by using the Gao-binge (Chronic+binge) protocol.22 As expected, although ethanol administration to mice significantly enhanced the serum alanine aminotransferase levels as well as serum levels of TNF-α, Ad–miR-217 injection exacerbated the ethanol-mediated effects (Supplemental Figure S4, B and C). Remarkably, Ad–anti-miR-217 injection completely blocked elevation of serum alanine aminotransferase and largely abolished the increased serum TNF-α in ethanol-administrated mice, suggesting that miR-217 indeed plays a crucial role in inflammation and liver hepatitis induced by ethanol in mice (Supplemental Figure S4). Paradoxically, the liver triglyceride or cholesterol levels were not significantly altered by Ad–miR-217 or Ad–anti-miR-217 injection in the setting of ethanol feeding in mice (data not shown).
Ad-Mediated Elevation of miR-217 in Mouse Livers Perturbs SIRT1–Lipin-1 Signaling
Consistent with the findings that miR-217 disrupts the SIRT1–lipin-1 axis in hepatocytes,12 we found that SIRT1 mRNA and protein expression were both significantly reduced in the livers of mice receiving Ad-miR-217 compared with Ad-GFP controls (Figure 7, A and B). Conversely, reduction of miR-217 abundance with Ad–anti-miR-217 increased SIRT1 mRNA and protein expression (Figure 7, A and B). Overexpression of Ad-miR-217 significantly induced total lipin-1 and lipin-1β mRNA expression, whereas mRNA abundance of human transformer-2-beta gene and lipin-1α mRNA were substantially reduced in mice receiving Ad-miR-217 (Figure 7C). Consistent with these observations, hepatic Lpin1b/Lpin1a ratio was increased by nearly sixfold in mice injected with Ad-miR-217 compared with controls (Figure 7D). However, no alterations in hepatic lipin-1 signaling were observed in mice that received Ad–anti-miR-217 or Ad-GFP.
Figure 7.
Ad-mediated elevation of miR-217 in the liver impairs SIRT1 signaling in mice. Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) were administered to 10- to 12-week-old male C57BL/6J mice twice on day 1 and day 5. After a 10-day period, mice were sacrificed. A: Relative hepatic mRNA levels of SIRT1. B: Liver extracts of mice infected with Ad-GFP, Ad–miR-217, or Ad–anti-miR-217 were immunoblotted with a SIRT1 or a β-actin antibody. C: Relative hepatic mRNA levels of total lipin-1, lipin-1α, lipin-1β, and SFRS10. D: Lpin1β/α ratio. Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; GFP, green fluorescent protein; Lpin1, lipin-1; SFRS10, human transformer-2-beta gene; SIRT1, sirtuin 1.
Discussion
Our previous work identified miR-217 as a regulator of ethanol-mediated excessive lipid accumulation in mouse hepatocytes.12 Here, we extend those observations by interrogating the role of miR-217 in regulating pathways of hepatic inflammation response after ethanol or LPS exposure. With the use of cultured RAW 264.7 macrophages or primary KCs, we demonstrate that LPS and ethanol each significantly up-regulated miR-217 abundance. More importantly, ethanol markedly augmented LPS-mediated miR-217 induction. We showed that overexpressison of miR-217 in macrophages promoted production of a panel of inflammatory cytokines stimulated by concurrent exposure to E+L. We further show that E+L-mediated increased expression of miR-217 in macrophages is functionally associated with attenuated expression of SIRT1 and disrupted SIRT1 signaling. Finally, Ad-mediated enrichment of hepatic miR-217 abundance induced hepatic steatosis and inflammation in vivo. Taken together, our findings suggest that the up-regulation of hepatic miR-217 by ethanol and LPS may play a vital role in hepatic inflammation (Figure 8).
Figure 8.
Proposed working model for the role of miR-217 in ethanol-induced inflammation response in Kupffer cells. The increased expression of miR-217 by ethanol, LPS, or combination of ethanol and LPS in Kupffer cells is functionally associated with disrupted SIRT1–AMPK–lipin-1α signaling leading to impairment of NF-KB and NFATc4 signaling pathways and generation of inflammatory cytokines. AMPK, AMP-activated kinase; LPS, lipopolysaccharide; NFATc4, nuclear factor of activated T cells c4; ROS, reactive oxygen species; SIRT1, sirtuin 1; TGF, transforming growth factor.
Our results demonstrate an intriguing dialogue between TGF-β and ROS signaling and miR-217 expression in macrophages exposed to E+L. We show that TGF-β and hydrogen peroxide induced miR-217 expression in a dose-dependent manner in macrophages, whereas miR-217 overexpression in macrophages significantly augmented ROS production and TGF-β expression with E+L. Because TGF-β is known to induce ROS generation,27 these observations imply that increased miR-217 expression in response to E+L exposure is likely to involve ROS production via both TGF-β–dependent and –independent mechanisms.
TGF-β is a major fibrogenic cytokine implicated in chronic liver disease progression in liver injury.28–30 Hepatic TGF-β is also significantly up-regulated in animal or human ALD.31–35 However, the role of TGF-β signaling in the pathogenesis of the early stage of liver diseases such as alcoholic steatohepatitis is not fully understood. Consistent with the findings that TGF-β up-regulates miR-217 expression,27 here, we show that TGF-β expression was significantly up-regulated in the macrophages exposed to E+L. In addition, we show that miR-217 not only stimulates TGF-β expression but also exacerbates E+L-mediated increases in TGF-β. Accordingly, it is tempting to speculate that miR-217 may up-regulate TGF-β both directly and indirectly through a positive feedback mechanism.
Another finding of the present study is the involvement of NFATc4 in mediating the proinflammatory effects of miR-217 or E+L in macrophages. Similar to NF-κB, NFATc4 plays an important role in the activation of inflammatory cytokines such as TNF-α and IL-6.21,26 The accumulation of NFATc4 in the nucleus leads to NFATc4 activation.21,26 We recently found that, in the liver, SIRT1 and NFATc4 are physically associated, and SIRT1 inhibits NFATc4 activity (M.Y., unpublished data).6 These findings together support the interpretation that E+L-mediated disruption of miR-217–SIRT1 axis may lead to activation of NFATc4 via modulation of NFATc4 acetylation/de-acetylation. Our findings further suggest that NF-κB and NFATc4 are likely to be coordinately regulated by the miR-217–SIRT1 axis. These two inflammatory regulators may then cooperate to regulate the expression of downstream proinflammatory cytokines induced by ethanol, LPS, or E+L in macrophages.
Emerging evidence suggests that lipin-1 plays a vital role in regulation of inflammation.21,35 Lipin-1 directly interacts with NFATc4 to inhibit NFATc4 transcriptional activity in adipocytes, which, in turn, inhibits inflammation.35 Two major lipin-1 protein isoforms, lipin-1α and lipin-1β, are derived from LPIN1 alternative mRNA splicing.6,35 The present findings suggest that lipin-1α but not lipin-1β exerts anti-inflammatory actions through NFATc4 inhibition in macrophages exposed to E+L or stimulated by miR-217. A more precise delineation of the role of lipin-1α in regulation of NFATc4 activity and inflammatory processes in response to miR-217, ethanol, and LPS in macrophages is currently under investigation.
The novelty of our present study lies in providing strong evidence that the miR-217–SIRT1–lipin-1 axis represents a pivotal signaling route in macrophage-dependent inflammation in response to ethanol and LPS. In addition, the observations that Ad-mediated overexpression of hepatic miR-217 promotes development and progression of steatohepatitis suggest that strategies to inhibit hepatic miR-217 could be a viable approach in attenuating both alcoholic and nonalcoholic steatohepatitis.
Acknowledgments
The following were kind gifts: NF-κB-responsive reporter-3xκB luciferase plasmid and wild-type SIRT1 (SIRT1wt) expression plasmids from Dr. Marty W. Mayo (University of Virginia); pcDNA 6.2-GW/miR-217 and pcDNA 6.2-GW/scmiR-217 expression plasmids from Dr. Gregory M. Vercellotti (University of Minnesota); and Ad-lipin-1α from Dr. Brian Finck (Washington University School of Medicine).
Footnotes
Supported by the NIH National Institute on Alcoholism and Alcohol Abuse grants AA015951 and AA013623 (M.Y.) and NIH National Institute of Diabetes and Digestive and Kidney Diseases grants DK06260 and DK052574 (N.O.D).
Disclosures: None declared.
Supplemental Data
miR-217 promotes expression of genes encoding inflammatory cytokines in RAW 264.7 macrophages exposed to E+L. RAW 264.7 cells were transfected with plasmids of control vector, miR-217, or Sc–miR-217. RAW264.1 cells were then treated with E+L for 18 hours. A: RT-qPCR was used to estimate relative mRNA levels of IL-1β. B: The relative mRNA levels of MCP-1 in RAW 264.7 cells transfected with plasmids of control, miR-217, and Sc–miR-217 treated with or without E+L. C: The relative mRNA levels of IFN-γ in RAW 264.7 cells transfected with plasmids of control, or miR-217, Sc–miR-217 treated with or without E+L. D: The relative mRNA levels of iNOS in RAW 264.7 cells transfected with plasmids of control, miR-217, or Sc–miR-217 treated with or without E+L. Data are expressed as means ± SEM. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. E+L, 50 mmol/L ethanol and 100 ng/mL lipopolysaccharide; IFN, interferon; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemotactic protein 1; RT-qPCR, quantitative real-time RT-PCR.
miR-217 exacerbates impairment of SIRT1 induced by the combination of E+L in RAW 264.7 macrophages. Cell extracts of RAW 264.7 macrophages transfected with plasmids of control vector, miR-217, Sc–miR-217, or Anti-miR-217 with or without E+L were immunoblotted with a SIRT1, p-AMPKα, AMPKα, or β-actin antibody. Relative SIRT1 protein expression (A and C). Relative levels of p-AMPKα/AMPKα ratio (B and D). E: Relative liver miRNA levels. Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. AMPKα, AMP-activated kinase α; E+L, ethanol and lipopolysaccharide; p-AMPKα, phosphorylated AMPKα; SIRT1, sirtuin 1.
miR-217 promotes activation of both NF-κB and NFATc4 in macrophages exposed to E+L. A: RAW 264.7 cells were transiently transfected with 10 μg of NF-κB–responsive reporter-3xκB luciferase and expression plasmids (5 μg for each) of miR-217 or sc-miR-217 and β-galactosidase (2 μg; internal control). E+L was added for 18 hours. At 48 hours after transfection, cells were harvested, and luciferase and β-galactosidase activities were determined. B: Relative protein levels of NFATc4 in Nuc or Cyto of RAW 264.7 macrophages transfected with plasmids of control vector miR-217 treated with or without E+L. C: Medium levels of TNF-α in RAW 264.7 cells infected with Ad-GFP, Ad-miR-217, lipin-1α treated with or without E+L. D: Medium levels of IL-6 in RAW 264.7 cells infected with Ad-GFP, Ad-miR-217, lipin-1α treated with or without E+L. Data are expressed as means ± SD. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. Ad, adenovirus; Cyto, cytoplasm; E+L, ethanol and lipopolysaccharide; GFP, green fluorescent protein; NFATc4, nuclear factor of activated T cells c4; Nuc, nucleus; TNF, tumor necrosis factor.
Ad-mediated elevation of miR-217 in the liver causes steatosis and provokes inflammation in mice. During the 10-day Gao-binge (Chronic+binge) ethanol treatment period, Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) was given to male C57BL/6J mice (10- to 12-week-old) twice on day 1 and day 5. Shown are relative levels of hepatic miR-217, miR-33, or miR-34a (A); serum ALT levels (B); and serum TNF-α (C). Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; ALT, alanine aminotransferase; EtOH, ethanol; GFP, green fluorescent protein; TNF, tumor necrosis factor.
Ad-mediated elevation of miR-217 in the liver promotes expression of genes encoding inflammatory cytokines in mice. During the 10-day Gao-binge (Chronic+binge) ethanol treatment period, Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) was given to male C57BL/6J mice (10- to 12-week-old) twice on day 1 and day 5. A: Relative mRNA levels of SAA1. B: Relative mRNA levels of LCN2. Dara are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; GFP, green fluorescent protein; LCN2, lipocalin 2; SAA1, serum amyloid A1.
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Associated Data
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Supplementary Materials
miR-217 promotes expression of genes encoding inflammatory cytokines in RAW 264.7 macrophages exposed to E+L. RAW 264.7 cells were transfected with plasmids of control vector, miR-217, or Sc–miR-217. RAW264.1 cells were then treated with E+L for 18 hours. A: RT-qPCR was used to estimate relative mRNA levels of IL-1β. B: The relative mRNA levels of MCP-1 in RAW 264.7 cells transfected with plasmids of control, miR-217, and Sc–miR-217 treated with or without E+L. C: The relative mRNA levels of IFN-γ in RAW 264.7 cells transfected with plasmids of control, or miR-217, Sc–miR-217 treated with or without E+L. D: The relative mRNA levels of iNOS in RAW 264.7 cells transfected with plasmids of control, miR-217, or Sc–miR-217 treated with or without E+L. Data are expressed as means ± SEM. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. E+L, 50 mmol/L ethanol and 100 ng/mL lipopolysaccharide; IFN, interferon; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemotactic protein 1; RT-qPCR, quantitative real-time RT-PCR.
miR-217 exacerbates impairment of SIRT1 induced by the combination of E+L in RAW 264.7 macrophages. Cell extracts of RAW 264.7 macrophages transfected with plasmids of control vector, miR-217, Sc–miR-217, or Anti-miR-217 with or without E+L were immunoblotted with a SIRT1, p-AMPKα, AMPKα, or β-actin antibody. Relative SIRT1 protein expression (A and C). Relative levels of p-AMPKα/AMPKα ratio (B and D). E: Relative liver miRNA levels. Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. AMPKα, AMP-activated kinase α; E+L, ethanol and lipopolysaccharide; p-AMPKα, phosphorylated AMPKα; SIRT1, sirtuin 1.
miR-217 promotes activation of both NF-κB and NFATc4 in macrophages exposed to E+L. A: RAW 264.7 cells were transiently transfected with 10 μg of NF-κB–responsive reporter-3xκB luciferase and expression plasmids (5 μg for each) of miR-217 or sc-miR-217 and β-galactosidase (2 μg; internal control). E+L was added for 18 hours. At 48 hours after transfection, cells were harvested, and luciferase and β-galactosidase activities were determined. B: Relative protein levels of NFATc4 in Nuc or Cyto of RAW 264.7 macrophages transfected with plasmids of control vector miR-217 treated with or without E+L. C: Medium levels of TNF-α in RAW 264.7 cells infected with Ad-GFP, Ad-miR-217, lipin-1α treated with or without E+L. D: Medium levels of IL-6 in RAW 264.7 cells infected with Ad-GFP, Ad-miR-217, lipin-1α treated with or without E+L. Data are expressed as means ± SD. n = 3 to 5 experiments. Means without a common letter differ, P < 0.05. Ad, adenovirus; Cyto, cytoplasm; E+L, ethanol and lipopolysaccharide; GFP, green fluorescent protein; NFATc4, nuclear factor of activated T cells c4; Nuc, nucleus; TNF, tumor necrosis factor.
Ad-mediated elevation of miR-217 in the liver causes steatosis and provokes inflammation in mice. During the 10-day Gao-binge (Chronic+binge) ethanol treatment period, Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) was given to male C57BL/6J mice (10- to 12-week-old) twice on day 1 and day 5. Shown are relative levels of hepatic miR-217, miR-33, or miR-34a (A); serum ALT levels (B); and serum TNF-α (C). Data are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; ALT, alanine aminotransferase; EtOH, ethanol; GFP, green fluorescent protein; TNF, tumor necrosis factor.
Ad-mediated elevation of miR-217 in the liver promotes expression of genes encoding inflammatory cytokines in mice. During the 10-day Gao-binge (Chronic+binge) ethanol treatment period, Ad-miR-217 or Ad–anti-miR-217 (0.5 to 1.0 × 109 active viral particle in 200 μL of phosphate-buffered saline) was given to male C57BL/6J mice (10- to 12-week-old) twice on day 1 and day 5. A: Relative mRNA levels of SAA1. B: Relative mRNA levels of LCN2. Dara are expressed as means ± SEM. n = 4 to 6 mice. Means without a common letter differ, P < 0.05. Ad, adenovirus; GFP, green fluorescent protein; LCN2, lipocalin 2; SAA1, serum amyloid A1.