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Published as doi: 10.1096/fj.06-6084fje.
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(The FASEB Journal. 2006;20:2112-2114.)
© 2006 FASEB

IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression

Johnson Rajasingh, Evelyn Bord, Corinne Luedemann, Jun Asai, Hiromichi Hamada, Tina Thorne, Gangjian Qin, David Goukassian, Yan Zhu, Douglas W. Losordo and Raj Kishore1

Division of Cardiovascular Research, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA

1Correspondence: Caritas St. Elizabeth’s Medical Center, Division of Cardiovascular Research, 736 Cambridge St., Boston, MA 02135 USA. E-mail: raj.kishore{at}tufts.edu

ABSTRACT

Inflammation plays an essential role in vascular injury and repair. Mononuclear phagocytes are important contributors in these processes, in part, via adhesive interactions and secretion of proinflammatory cytokines. The antiinflammatory cytokine interleukin (IL)-10 suppresses such responses via deactivation of monocytes/macrophages and repression of inflammatory cytokine expression. The mechanisms of IL-10’s suppressive action are, however, incompletely characterized. Here, we report that systemic IL-10 treatment after carotid artery denudation in mice blunts inflammatory cell infiltration and arterial tumor necrosis factor (TNF) expression. At the molecular level, in a human monocytic cell line, U937 IL-10 suppressed LPS-induced mRNA expression of a number of inflammatory cytokines, mainly via posttranscriptional mRNA destabilization. Detailed studies on IL-10 regulation of TNF-{alpha} mRNA expression identified AU-rich elements (ARE) in the 3' untranslated region as a necessary determinant of IL-10-mediated TNF-{alpha} mRNA destabilization. IL-10 sensitivity to TNF depends on the ability of IL-10 to inhibit the expression and mRNA-stabilizing protein HuR and via IL-10 mediated repression of p38 mitogen-activated protein (MAP) kinase activation. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.—Johnson Rajasingh, Evelyn Bord, Corinne Luedemann, Jun Asai, Hiromichi Hamada, Tina Thorne, Gangjian Qin, David Goukassian, Yan Zhu, Douglas W. Losordo, and Raj Kishore. IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression.


Key Words: mRNA stability • AU-rich elements

THE ASSOCIATION OF inflammation with atherosclerosis and restenosis is now fairly well established. Restenosis, a persistent complication of percutaneous vascular interventions, is thought to be a complex response to injury, which includes early thrombus formation, neointimal growth, and acute inflammation. Mononuclear phagocytes are likely participants in the host response to vascular injury, via accumulation, infiltration, and adhesion to injured arteries and via the secretion of proinflammatory cytokines and chemokines (1 2 3) . In this context, TNF-{alpha} (hereto referred as TNF), produced largely by activated monocytes/macrophages, is known to be negatively associated with restenosis and atherosclerosis (1 , 4 5 6 7) . We and others have previously shown that TNF represses re-endothelializaion, inhibits endothelial cell (EC) proliferation, and is a strong mitogen for vascular smooth muscle cell (VSMC) proliferation (6 7 8 9) . Thus regulation of TNF production via deactivation of inflammatory cells within revascularized arteries could represent a desirable approach for restenosis prevention.

IL-10 (interleukin-10), a potent antiinflammatory cytokine is a strong deactivator of monocytes and suppressor of the synthesis of TNF and other proinflammatory cytokines (10 , 11) . Interestingly, IL-10 knockout mice exhibit unregulated inflammatory activity exemplified by enhanced TNF accumulation, which is associated with a variety of pathogenic outcomes, including endotoxemia, intestinal inflammation, and atherosclerosis (12 13 14) . However, the molecular mechanisms and signaling events that govern IL-10 inhibition of inflammatory gene expression are poorly understood and remain to be fully characterized. Here, we report that systemic IL-10 treatment of mice following carotid artery denudation blunts the inflammatory cell infiltration and local TNF expression. At the molecular level, IL-10 strongly inhibits LPS-induced mRNA expression of TNF in human monocytic cell line, U937, primarily via reduction in the TNF mRNA stability. Furthermore, we report that IL-10 regulation of TNF mRNA stability depends on AU-rich elements (ARE) present in the 3' untranslated region (UTR) of TNF mRNA and that IL-10 sensitivity to TNF depends on the ability of IL-10 to inhibit the LPS activation of p38 MAP kinase and via IL-10- mediated suppression of mRNA-stabilizing protein, HuR.

MATERIALS AND METHODS

Animals and carotid artery denudation
Carotid injury was performed in 20 C57/BL6 male mice of 4–6 wk of age, as reported previously (15) . All animal procedures were performed in accordance with Caritas St. Elizabeth’s Institutional Animal Care and Use Committee. Mice were divided into two groups of 10 each and were treated with either 50 µg/kg recombinant murine IL-10 (intraperitoneal injections)/alternate days or with nonimmune murine IgG protein as a control.

Histology
Arterial cross sections obtained on day 21 after injury from IL-10- and IgG-treated animals were stained with mouse anti-TNF antibodies and with macrophage/monocyte specific antibody (Ab), M4/80, to immunostain for inflammatory cells.

Cell culture and antibodies
U937 human monocyte/macrophage cell line was obtained from American Type Culture Collection (ATCC) and cultured in RPMI 1640 media supplemented with 10% FBS and antibiotics in 5% CO2 atmosphere. Cells were stimulated with LPS and or IL-10 at a dose of 10 ng/ml unless otherwise indicated. Thioglycollate-elicited, C57BL/6 murine peritoneal macrophages were isolated and cultured, as described elsewhere (16) . Anti-TNF antibodies were purchased from BD Pharmingen. Anti-HuR and anti-TTP antibodies were obtained from Santa Cruz Inc.(Santa Cruz, CA). Antibodies for phosphor- and total ERK1/2, JNK1, and p38 MAP kinases were purchased from Cell Signaling. Recombinant murine IL-10 was obtained from R&D Systems (Minneapolis, MN).

Constructs, TNF-chloroamphenicol acetyltransferase chimeras and transfections
TNF 5'UTR, coding region (CR) and 3'UTR fragments were generated by polymerase chain reaction (PCR) and were subcloned into pCAT3 control vector (Promega) at the XbaI site immediately downstream of the chloroamphenicol acetyltransferase (CAT) coding region (pCAT-TNF5'UTR, pCAT-TNFCR, and pCAT-TNF3'UTR). Expression of CAT in these vectors is driven by SV40 promoter/enhancer. A 70-nt (nt 419–489 of TNF 3'UTR) region, which contains both clustered AU pentamers and isolated pentamers was also subcloned into pCAT3 control vector (pCAT3 TNF3'UTRARE). In addition, another construct was prepared that contains similar TNF-ARE sequence with the exception that clustered AU pentamers between nt 454–76 were mutated by substitution (substituted nt underlined in Fig. 4 ; pCAT3 TNF3'UTRAREmut). The nucleotide sequences for cloned fragments were determined at Molecular Biotechnology Core Facility at Tufts University. All the generated chimeric constructs are shown in Fig. 4A . Cells were transiently transfected as described previously (9) .


Figure 1
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  		Figure 1. IL-10 treatments inhibits inflammatory cell infiltration and arterial TNF expression following carotid artery denudation. A) Representative pictures showing M4/80 immunostained inflammatory cells in carotid arteries obtained from mice treated with IgG or IL-10 28 days postinjury. B) Quantification of number of positive stained cells/visual field. C) Representative immunochemical staining showing TNF expression in 21 days postinjury carotid arteries obtained from IgG or IL-10 treated mice.


Figure 2
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  		Figure 2. IL-10 inhibits proinflammatory cytokine/ chemokine mRNA expression. U937 human monocytic cell line was treated with 10 ng/ml LPS for 1 h followed by IL-10 (10 ng/ml) for 1–2 h. Total RNA extracted was subjected to multiple cytokine/chemokine mRNA expression by RPA analysis using in vitro synthesized ribo-probe using multiprobe cDNA template and kits (Ambion). Representative autoradiograph from at least 3 similar experiments (A). B) mRNA expression of individual cytokine/chemokine was normalized to GAPDH expression and quantified,. and data are shown as graph *P < 0.001; UT, untreated).


Figure 3
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  		Figure 3. IL-10 suppresses monocyte gene expression through diverse mechanisms. A) Rate of de novo mRNA synthesis was determined in nuclear run-on experiments in U937 cells treated simultaneously with LPS and IL-10 for indicated time. IL-10 significantly inhibited IP-10 mRNA transcription without affecting the transcription of TNF, IL-1ß, and MCP-1 (*P<0.01; #P=ns). B) TNF and IP-10 mRNA stability was assessed in cells stimulated with LPS (10 ng/ml) for 1 h followed by IL-10 treatment (10 ng/ml) for indicated times with or without actinomycin D (5 µg/ml). mRNA expression at each time point (representative autoradiograph, top) was quantified by NIH image analysis and normalized to ß-actin control expression lower panel). IL-10 significantly reduced TNF mRNA half-life without affecting that of IP-10 (*P<0.01).


Figure 4
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  		Figure 4. Sequences within TNF3'UTR confer IL-10 sensitivity on CAT mRNA. A) Schematic representation of CAT-TNF chimeric constructs detailed in materials and methods. B) Constructs shown above were transiently transfected in U937 cells and were treated with IL-10 and actinomycin D as indicated. CAT mRNA half-life was determined by RPA using CAT DNA template (Promega) to synthesize antisense riboprobe. Remaining CAT mRNA (% of 0 time point) at 2 h post IL-10/Act D treatment from chimeric constructs transfected cells was compared to that of pCAT control transfected cells by densitometric analysis of autorads and plotted (a, d P<0.01; c P<0.001; b,e,f P<0.05).

Oligonucleotides and DNA and RNA elctrophoretic mobility shifts assays (EMSA)
DNA-EMSAs using consensus STAT3 oligonucleotides (Santa Cruz) and 5 µg of nuclear proteins from variously treated U937 cells were carried out as described previously (9 , 16 , 17) . For RNA-EMSAs, cytoplasmic extracts from variously treated U937 cells were allowed to react with a radio-labeled ([{alpha}-32P]-uridine triphosphate) single-stranded RNA fragment, corresponding to the sense orientation of the 3'UTRARE of the human TNF mRNA prepared by in vitro transcription (IVT). To generate the template for IVT reaction, TNF 3'UTRARE and TNF 3'UTRAREmut sequence were prepared in pBlueScript KS (pBS; Stratagene). pBSTNF3'UTRARE was linearized with Bam H1, whereas pBSTNF 3'UTRAREmut was cut with HindIII. One microgram of DNA was used for IVT. EMSAs were carried out in a 20-µl reaction containing 20 µg of cytoplasmic extract and 4 ng radiolabeled RNA probe in binding buffer (10 mM HEPES, 100 mM KCl, 3 mM MgCl2, 2 mM DTT, 5% glycerol) for 20 min at 4°C. Heparin (30 µg/ml) and yeast RNA (50 µg/ml) were added, and the mixture was incubated further for 10 min. In competition experiments, excess unlabeled RNA probe was added to the reaction before the addition of radiolabeled probe. RNA not associated with protein was digested with RNase T1 (30 U) for 20 min at room temperature. The RNase- resistant complexes were resolved by nondenaturing PAGE (6%), in Tris-Borate-EDTA buffer (TBE). Gels were dried and autoradiographed.

Nuclear run-on assays
Nuclear run-on experiments to measure nascent RNA transcripts were essentially performed as described before (9 , 17) .

RNA isolation and ribonuclease protection assay (rNase protection assay)
Total cellular RNA isolation and preparation of in vitro transcribed riboprobes and rNase protection assay (RPA) assays, as well as mRNA half-life assays were carried out as described before (9 , 17) .

Western blot analysis
Western blot analysis were performed essentially as described before (9 , 17) .

Statistical analyses
All experiments were carried out at least 3 or more times. Results are presented as means ± SEM. Comparisons were done by ANOVA (GB-STAT, Dynamic Microsystems Inc., Silver Spring, MD) or {chi}2 test for percentages. All tests were two-sided, and a P value less than 0.05 were considered statistically significant.

RESULTS

Systemic recombinant IL-10 therapy inhibits TNF expression and inflammatory cell infiltration in injured mouse carotid arteries.
We have previously shown that in vitro TNF inhibits endothelial cell proliferation by inducing cell cycle arrest, in part, by down-regulating cyclin A expression, and inducing apoptosis (9 , 17) . We have also reported that in a rat model of carotid denudation, TNF, expressed at the sites of arterial injury, inhibits re-endothelialization (ReEndo), resulting in increased intima: media ratios and that neutralization of TNF with TNF-soluble receptor significantly accelerates re-endothelialization and inhibits neointimal thickening (6) . Because mononuclear phagocytes are the major source of TNF at arterial injury and mediate injury induced arterial remodeling and because IL-10 acts as a potent deactivator of inflammatory cells, we examined the effect of systemic IL-10 treatment on inflammatory cell infiltration and local arterial TNF expression in a mouse model of carotid injury. Carotid injury was performed in 20 C57/BL6 mice, and mice were divided into two groups of 10 each and were treated with either 50 µg/kg recombinant murine IL-10 (i.p. injections)/alternate days or with nonimmune murine IgG protein as control. Arteries were harvested on day 21 postinjury, and the number of monocytes/macrophages was counted in the arterial cross sections. Sections were stained with macrophage/monocyte specific Ab, M4/80, and stained cells were counted by phase contrast microscopy in six different fields/artery by two blinded investigators. As shown in representative staining in Fig. 1 A and quantified in Fig. 1B , IL-10 treatment significantly reduced the number of infiltrating inflammatory cells at day 21. Reduction in number of infiltrating monocytes/macrophages was correlated to the strong immunoreactivity for TNF from arteries obtained from IgG-treated mice, while TNF expression was largely abrogated in arteries from mice treated with systemic IL-10 (Fig. 1C ).

IL-10 suppresses monocyte gene expression through diverse mechanisms
In the next series of experiments, we focused our attention on understanding the mechanistic and signaling events that may mediate IL-10 induced monocyte deactivation and TNF inhibition. Several studies on the inhibitory function of IL-10 in mononuclear phagocytes led to the general conclusion that IL-10 acts through a diverse collection of mechanisms, which include reductions in gene transcription and in the stability and/or translation of target mRNAs (10 , 11 , 18) . To evaluate the mechanisms of IL-10 action at the molecular level, U937 cells were stimulated with LPS alone for 1 h (10 ng/ml) or with LPS+interleukin-10 (10 ng/ml) for 1–2 h. RNA was extracted and was subjected to RPA analysis using a custom-made multiprobe human cytokine/chemokine cDNA templates to generate in vitro synthesized riboprobes. As shown in Fig. 2 A, B, LPS stimulation led to potent up-regulation of all the chemokine/cytokine genes that were assayed, and IL-10 suppressed the mRNA expression of all the genes except that of MIG and RANTES.

To gain a better understanding of mechanisms of IL-10’s suppressive action, rate of de novo transcription was assessed for TNF, IP-10, IL-1ß, and MCP-1 genes in nuclear run-on experiments (Fig. 3 A). These experiments indicated that LPS-induction of IP-10 gene is inhibited by IL-10 at the level of transcription. In contrast, LPS-induced transcription of TNF, IL-1ß, and MCP-1, though substantial, appears to be largely unaltered in the presence of IL-10, suggesting that IL-10 represses TNF at post-transcriptional level. To validate this notion, studies on the stability of specific mRNAs (TNF and IP-10) were conducted by examining the rate of decay following blockade of further transcription using actinomycin D. These experiments indicated that IL-10 can induce marked increase in TNF mRNA degradation, whereas no such effect was observed when IP-10 mRNA decay was evaluated (Fig. 3B ). IL-10 also decreased mRNA half-lives of IL-1ß, MCP-1 and IL-12p40 (data not shown). We also assessed the production of the TNF protein in cellular extracts, as well as TNF secreted into the culture medium using heparin-sepharose adsorption; SDS-PAGE and Western blot analysis with rabbit polyclonal antibody (pAb) specific for the TNF protein, which showed similar reduction of LPS-induced TNF protein by IL-10 (data not shown).

Sequences in TNF3'UTR confer instability to chloramphenicol acetyltransferase (chloroamphenicol acetyltransferase) mRNA.
To determine whether sequences in TNF mRNA that confer IL-10 sensitivity are able to impart IL-10-mediated mRNA destabilization of otherwise stable reporter gene, we utilized CAT-TNF chimeric constructs shown in Fig. 4 A. Constructs were generated as described in Methods section. We determined the efficacy of cloned TNF sequences to impart constitutive or IL-10-mediated mRNA instability to CAT reporter gene. U937 cells were transiently transfected with constructs shown in Figure 4A , stimulated or not with IL-10 and treated with ActD to prevent further transcription before RNA was isolated and analyzed for CAT mRNA levels by RPA using in vitro transcribed CAT riboprobe generated by using CAT cDNA template (Promega). CAT mRNA transcribed from pCAT control was highly stable over 2-h period of the experiments. Similarly, CAT mRNA half-life in constructs containing either 5'UTR or coding region (CR) of TNF was similar to that of pCAT control and was also insensitive to IL-10 treatment (Fig. 4B , black, white and gray bars). Insertion of TNF3'UTR, however, significantly reduced the intrinsic half-life of CAT mRNA (P<0.01a compared with pCAT control) that was further reduced in the presence of IL-10 (P<0.001c compared to pCAT control). Similar CAT mRNA decay profile was observed in cells transfected with pCAT3'UTRARE, however, not to the same extent as is the case with complete 3'UTR (P<0.05b and P<0.01d for intrinsic and IL-10-induced decay, respectively, compared to pCAT control). Interestingly, however, substitution mutations in pCAT3'UTRAREmut (disruption of reiterated cluster of ARE pentamers) resulted in substantially reduced rate of intrinsic CAT decay (P<0.05e compared to pCAT-TNFARE), as well as significant loss of IL-10 sensitivity (P<0.01f compared to pCAT-TNFARE). Taken together, these data suggest that ARE sequences between nt 454–476 in TNF 3'UTR are necessary for both intrinsic and IL-10-mediated mRNA degradation; however, a faster decay rate of the CAT mRNA containing intact 3'UTR suggests that ARE sequences between nt 454 and 476 are not fully sufficient for TNF mRNA degradation and that additional sequences in 3'UTR beyond defined ARE region may also cooperate in this process. Indeed, isolated ARE are located in TNF 3'UTR.

IL-10 inhibits HuR expression and binding to TNF-3'UTRARE sequences
To determine the modulations in the binding of protein/factors to TNF-ARE sequences, cytoplasmic extracts from variously treated U937 cells were made and allowed to react with a radio-labeled ([{alpha}-32P]-uridine triphosphate) single stranded RNA fragment corresponding to the sense orientation of the 3'UTRARE of the human TNF mRNA prepared by in vitro transcription (IVT) in RNA-EMSA experiments. TNF3'UTRARE formed 3 complexes with cytoplasmic extracts from untreated (UT, lane 1) cells, of which complex II was subject to up-regulation by LPS (lane 2). IL-10 reduced the formation of both complex I and to a greater extent that of complex II without affecting complex III (lanes 3 and 4; Fig. 5 A). Both of these complexes represented specific binding activities to TNF-ARE, as competition with excess cold probe efficiently competed with the complex formation by labeled probe (lanes 5 and 6). Complex II appears to be specific to intact overlapping ARE pentamers, as disruption of overlapping UA pentamers (TNF3'UTRAREmut; Fig. 4A ) failed to compete with complex II formation even at 50-fold excess amount (lane 8). Inclusion of antibodies to RNA stabilizing protein HuR, a member of elav-like family of RNA-binding proteins, in the reaction mix before the addition of labeled probe "supershifted" complex II from LPS-treated extracts without affecting complexes I and III (Fig. 5B ). Another known TNF-ARE binding protein, tristetraprolin (TTP), which is implicated in controlling intrinsic TNF mRNA degradation, competed with complex III formation, indicating that it is a constituent of complex III. However, IL-10 had no effect on the complex III, suggesting TTP is insensitive to IL-10 (Fig. 5B ). These data implied that IL-10 might modulate the expression of proteins/transfactors that may bind to TNF-ARE and act to stabilize TNF mRNA. To test this, protein expression of HuR and TTP was examined in U937 whole cell extracts. As shown in Fig. 5C , HuR expression was substantially reduced by IL-10, while TTP expression had no effect, suggesting that TNF mRNA destabilization by IL-10 may partially result from IL-10-mediated inhibition of HuR.


Figure 5
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  		Figure 5. IL-10 inhibits HuR binding to TNF-3'UTR ARE sequences in RNA EMSAs. A) Representative RNA-EMSA. Cytoplasmic extracts from variously treated U937 cells were made and allowed to react with a radio-labeled ([{alpha}-32P]-uridine triphosphate) single-stranded RNA fragment corresponding to the sense orientation of the 3'UTRARE of the human TNF mRNA prepared by in vitro transcription (IVT). For competition, indicated fold excess of unlabeled IVT probe were added 20 min prior to the addition of radioactive probe (Lanes: 1, Untreated; 2, LPS; 3, interleukin-10; 4, LPS+interleukin-10; 5, LPS+25X, 3'UTRARE cold probe; 6, LPS+50x3'UTRARE cold probe; 7, LPS+25x3'UTRAREmut cold probe; and 8, LPS+50x3'UTRAREmut cold probe). B). For super shift of observed complexes, anti-HuR, anti-TTP antibodies, and control IgG antibodies were mixed in the EMSA reaction before the addition of radiolabeled probe. Anti-HuR supershifted IL-10-sensitive complex II, whereas anti TTP diminished IL-10 insensitive complex III. C) Total protein extracts from cells treated as indicated were subjected to Western blot analysis for known ARE binding proteins, HuR, and TTP. IL-10 suppressed LPS-induced expression of HuR.

SiRNA-mediated HuR silencing abrogates the effect of IL-10 on TNF mRNA destabilization
Because data shown in Fig. 5 suggest HuR as a target TNF-ARE binding protein, which is sensitive to IL-10 and because HuR is a known ARE-binding protein that acts to stabilize a given mRNA, we performed additional experiments to determine whether inhibition of HuR in the intact cells will affect TNF mRNA stability in the absence and presence of IL-10 treatment. Specific human HuR small interfering RNA (siRNA) and a scrambled control siRNA were purchased from Ambion Inc.(Austin, TX). The HuR siRNA duplex sequence used in the experiment was as follows. HuRSi: 5' AAGCCUGUU CAGCA GCAUUGG-3', which targets to position 111 to 131 of nucleotides relative to the start codon of HuR mRNA. U937 cells were transfected in duplicate dishes with either HuR siRNA or scrambled siRNA (0.1 ìM) by using the Lipofectamine 2000 and Lipofectamine Plus reagents (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Twenty-four hours later, the medium was changed with fresh growth medium containing LPS for indicated times. Total protein extracts from transfected and treated cells were analyzed for the knock-down of HuR. As shown in Fig. 6 A, HuR siRNA transfection completely abrogated HuR protein expression, whereas there was no inhibition of HuR by scrambled siRNA. Both HuR and control siRNA transfected cells were stimulated with LPS and LPS+interleukin-10 for 2 h and treated with ActD for indicated times. RNA extracted from siRNA-transfected cells was analyzed by RPAs to determine the TNF mRNA half-life. As shown in Fig. 6B , TNF mRNA decay in scrambled siRNA transfected cells (left side) was similar to that observed in untreated and IL-10 treated untransfected cells (Fig. 3B ) shown in previous sections. However, knock down of HuR by specific siRNA significantly reduced intrinsic TNF mRNA decay rate (P<0.01, LPS/ActD 2h in scrambled siRNA transfected cells vs. LPS/ActD 2h in scrambled siRNA transfected cells). Moreover, silencing of HuR completely abrogated IL-10-mediated enhanced degradation of TNF mRNA shown in previous sections (Fig. 3B ).


Figure 6
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  		Figure 6. siRNA-mediated HuR silencing abrogates the effect of IL-10 on TNF mRNA destabilization. A) Western blot analysis showing the specificity of HuRsiRNA inhibition of HuR protein expression. B) Quantification of TNF mRNA stability experiments in cells transfected with HuRsiRNA and scrambled control siRNA. Silencing of HuR abrogates the effect of IL-10 on enhanced TNF mRNA decay.

IL-10-mediated TNF mRNA instability is partly mediated by p38 MAP kinase
Recent data from several laboratories indicates the participation of members of MAP kinase family in the determination of mRNA stability/translation of several genes (19 20 21 22 23 24) . However, with regard to IL-10-mediated regulation of mRNA stability/translation, previous reports have provided controversial results on the effect of IL-10 on MAPK activation (25 26 27) . We therefore examined the influence of IL-10 on LPS-induced activation of all three members of MAP kinase family. U937 cells were treated with LPS with or without IL-10 for 15–60 min and phosphorylation of ERK1/2, JNK1, and p38 MAP kinases was determined by Western blot analysis using specific antibodies (Cell Signaling). Same blots were stripped and reprobed for the protein expression of respective total kinase expression. As shown in Fig. 7 A, IL-10 had no effect on the LPS-induced activation of either ERK1/2 or JNK1 MAP kinases. Activation of LPS-induced p38 MAP kinase, however, was strongly inhibited by IL-10 at all time points studied (Fig. 7B ). To investigate if IL-10-mediated suppression of p38MAPK is involved in TNF mRNA instability, U937 cells were transfected with a constitutively active p38MAPK expression vector (pcDNA3-CAp38) or corresponding empty vector. A subset of untransfected cells was used as a control. Transfected cells were treated with LPS or LPS+ IL-10 and ActD for 1 h, and RNA was used to determine the remaining TNF mRNA compared to basal TNF level after 1 h LPS stimulation. As shown in Fig. 7C , overexpression of constitutively active p38MAPK significantly reversed intrinsic (P<0.05), as well as IL-10-induced (P<0.01) TNF mRNA destabilization, compared to untransfected or empty vector-transfected cells.


Figure 7
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  		Figure 7. IL-10-dependent inhibition of TNF mRNA instability is mediated by p38 MAP kinase. A) Protein extracts from LPS and LPS+interleukin-10-treated U937 cells were analyzed for the phosphorylation of MAPK family members by Western blot analyses using specific antibodies. B) Quantification of the effect of IL-10 on p38MAPK phosphorylation. C) Cells were transiently transfected with constitutively active p38MAPK (pcDNA3-CAp38) or corresponding empty vector (pcDNA3-EV) and were treated or not with IL-10 and Act D as indicated before TNF mRNA stability was determined by RPAs. Bars represent quantification of TNF half-life in 3 similar experiments.

Finally, to demonstrate that these findings are representative of primary cells and not only transformed cells, we confirmed some of the experiments described in preceding sections in primary mouse peritoneal macrophages. Like U937 cells, IL-10 substantially reduced LPS-induced TNF mRNA expression (Fig. 8 A). Nuclear run-on experiments in mouse peritoneal macrophages treated with LPS (10 ng) or LPS+interleukin-10 (10 ng each) for 15–120 min exhibited rates of de novo TNF mRNA synthesis similar to U937 cells, suggesting that IL-10 does not inhibit TNF transcription in primary mouse macrophages (Fig. 8B ). IL-10 treatment of mouse peritoneal macrophages reduced TNF mRNA stability at all time points (P<0.01), as evident from the actinomycin D chase experiments (Fig. 8C ). Additionally, as shown in Fig. 8D , IL-10 also suppressed LPS-induced activation of p38 MAP kinase. Taken together, these observations suggest that irrespective of primary or transformed cells, IL-10-mediated suppression of TNF expression involves p38 MAP kinase inhibition-mediated TNF mRNA destabilization.


Figure 8
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  		Figure 8. IL-10 suppression of TNF expression in U937 cells is mimicked in primary mouse macrophages. A) IL-10 suppresses LPS-induced TNF expression in peritoneal mouse macrophages. Quantification of 3 RPA experiments is shown. B) Rate of de novo TNF mRNA synthesis was determined in nuclear run-on experiments in mouse peritoneal macrophages treated simultaneously with LPS and LPS+interleukin-10 for indicated time. IL-10 treatment did not alter the LPS-induced transcription of TNF (P =ns). C) TNF mRNA stability was assessed in cells stimulated with LPS (10 ng/ml) for 1 h followed by IL-10 treatment (10 ng/ml) for indicated times with or without actinomycin D (5 µg/ml) by RPAs. mRNA expression at each time point was quantified by NIH image analysis and normalized to control ß-actin mRNA expression. IL-10 significantly reduced TNF mRNA half-life (*P<0.01). D) Protein extracts from LPS and LPS+interleukin-10 treated mouse peritoneal macrophages were analyzed for the phosphorylation of p38 MAPK by Western blot analyses using specific antibodies. Each experiment was repeated for at least 3 times.

DISCUSSION

The multifactorial pathophysiology of restenosis remains as yet not entirely defined, however, experimental and clinical data indicate that inflammation is of major importance to the restenotic process. Inflammatory cells are activated promptly after vascular injury and recruited to the site of injury and release mediators such as TNF and other proinflammatory cytokines that facilitate VSMC migration and proliferation on one hand and inhibition of EC proliferation and re-endothelialization on the other (1 2 3 , 6 , 9 , 28) . Monocytes have been hypothesized to serve as markers, initiators, and promoters of arterial occlusive disease. In particular, activated monocytes may contribute to neointimal thickening (1 , 2) through the production of proinflammatory cytokines and chemotactic factors, which activates other effector cells like VSMC and EC, facilitating accumulation, infiltration, and adhesion of monocytes to these cells. Therefore, control of postinjury inflammatory response represents a desirable approach to curb restenosis. Justifiably, much emphasis has been placed on the mechanisms involved in enhanced expression of gene products important in the development of inflammatory function in the arterial microenvironment. Lately, however, information on the action and impact of agents that suppress inducible gene expression has begun to emerge. The control of potential tissue damage and vascular remodeling that frequently accompanies inflammation is of obvious importance. In addition, because most thoroughly studied stimuli such as TNF, which is the focus of this study, are known to act on most cell types, antiinflammatory agents that exhibit restricted cell type specificity may also be important determinants of cell type-specific patterns of gene expression in vivo.

IL-10, originally described as an inhibitor of cytokine synthesis exhibiting a broad spectrum of suppressive activity is now recognized as an important regulator of homeostasis with respect to the inflammatory status of the whole organism. It has emerged as a macrophage deactivator competent to suppress the expression of inflammatory mediators, as well as the macrophages’ ability to support accessory functions to adaptive immunity (10) . Mice in which the IL-10 gene has been deleted by gene targeting exhibit unregulated inflammatory activity exemplified by enhanced TNF accumulation and which is associated with a variety of pathogenic outcomes, including atherosclerosis (12 13 14) . Information regarding the antiinflammatory effects of IL-10 to the neointimal thickening and restenosis after arterial injury is limited and has only begun to emerge lately. Recent studies in various animal models have suggested that IL-10 plays an important role in the suppression of intimal hyperplasia and restenosis and atherosclerosis (29 30 31 32) . None of these studies, however, looked either at the specific inflammatory gene repression in response to IL-10 or the mechanisms of such inhibition thus necessitating further mechanistic studies. Our data presented in this study thus not only demonstrate physiological in vivo effect of IL-10 therapy in terms of reduced TNF expression and inflammatory cell infiltration, but also details the mechanistic insights into IL-10 suppressive action using TNF as a model gene.

Considering the pleiotropic effects of TNF on various cell types, it is not surprising that regulation of TNF occurs at multiple levels, including transcription, mRNA stability, and translation (33 34 35) . Because monocyte activation and proinflammatory cytokine secretion (such as that of TNF) have been shown to be negatively associated with atherosclerosis and restenosis, therefore, understanding of TNF biosynthetic regulation in monocytes is a key step for the development of new strategies to treat these diseases. Modulation of mRNA stability is an important mechanism of TNF biosynthesis. Although the mechanisms involved in post-transcriptional gene regulation are complex, the mRNA itself contains sequence-specific information that determines its stability (33 , 36) . It is now fairly well established that the key element involved in the mRNA stability and translational regulation is the ARE located in the 3' untranslated region (UTR) of labile genes, including TNF mRNA (33 , 36) . Within TNF 3'UTR, a 70-nucleotide-long sequence containing AREs has been shown to play a major role in the post-transcriptional control of TNF mRNA, although mechanisms of regulation have mostly been described at the level of translation (37 , 38) . The most compelling evidence for AREs as the regulators of TNF biosynthesis comes from an in vivo study that addressed the impact of deleting TNF-ARE from mouse genome on the regulation of TNF biosynthesis and on the physiology of the host. It revealed that the absence of the ARE affects mechanisms responsible for TNF mRNA instability and translational repression resulting in robust TNF accumulation and the development of chronic inflammatory arthritis and Crohn’s-like inflammatory bowel disease (37) . Although this report clearly confirmed the physiological importance of the ARE for the proper regulation of TNF production, much however, remains to be understood concerning the ARE-mediated mechanisms controlling mRNA stability and translation. Moreover, in some situations, ARE-containing mRNAs can be stabilized, indicating that ARE-mediated instability can be modulated (10 , 11 , 22 , 36) . Thus it appears that AREs can mediate destabilization and stabilization, as well as translation of certain mRNAs largely in a stimulus/gene-specific manner, depending on the modulations in transfactors/proteins that bind to AREs.

Few ARE-binding proteins purported to modulate TNF regulations have been described. These include, TIA, TIAR, TTP, and HuR. While TIA and TIAR have been implicated in the translational control of TNF mRNA, TTP is known as TNF mRNA destabilizing protein, whereas HuR on binding to AREs stabilizes TNF mRNA (39 40 41 42) . Whether IL-10 modulates the binding kinetics, expression, or function of these TNF-ARE binding proteins is, however, not elucidated. Our results thus provide a novel finding that IL-10 treatment inhibits the expression of HuR and represses the binding of HuR to TNF ARE, resulting in the enhanced degradation of TNF mRNA. Interestingly, IL-10 has no effect on the expression and function of TNF mRNA-destabilizing protein, TTP.

The signaling mechanisms involved in IL-10-mediated TNF mRNA decay are poorly understood. Our data, therefore, attempt to fill these gaps by providing systematic analyses of not only the sequence dependence for IL-10 suppressive action but also IL-10 inhibition of p38 MAPK activation as a potential mechanism for IL-10-mediated TNF mRNA decay. Data from several laboratories indicate the participation of members of MAP kinase family in mRNA stability/translation of several genes. We have previously demonstrated that chronic ethanol-induced stabilization of TNF mRNA is p38MAPK dependent (22) . Other studies also pointed toward a role of p38 in the modulation of TNF mRNA stability (23 , 24) . However, with regard to IL-10-mediated regulation of mRNA stability/translation, previous reports have provided controversial results on the effect of IL-10 on MAPK activation (25 26 27) . Although data reported by Denys et al. (26) showed no involvement of p38 MAP kinase in IL-10 inhibition of TNF, data reported by Kontoyiannis et al. (27) demonstrated that IL-10 modulates ARE-dependent TNF mRNA translation via suppressing the activation of p38 MAPK, without affecting mRNA stability. The reduction in the LPS-induced p38 phosphorylation by IL-10 in our studies would therefore be in agreement with the latter study; however, in contrast to this study we did find the evidence that IL-10 inactivation of p38 is linked to the destabilization of TNF mRNA. It should be noted that cell-type specificity (primary vs. cell lines, mouse vs. human cells), IL-10 treatment modalities and experimental procedures might also explain some of the disparate results. For example, in contrast to our studies, Denys, et al. (26) measured p38 MAP kinase activation by measuring kinase activities rather than phosphorylation status; unlike our studies, there were no negative controls for specificity (either chemical inhibitors or genetic overexpression approaches), and pretreatment with IL-10 followed by short LPS exposure (20 min) rather than measurement of LPS-induced p38 MAP kinase activation in cells simultaneously treated with IL-10. Our data would also indicate that IL-10 acts upstream of p38 activation. It has been shown that IL-10 still inhibits TNF production in MKK3-deficient macrophages despite reduction in p38 phosphorylation (43) . This suggests that MKK3 is not required, and the most likely alternative could be that IL-10 interferes with MKK6 signals activating p38. This, however, remains to be confirmed in our studies.

In summary, because IL-10 function and signaling are important components for control of inflammatory responses, our data attempt to provide a better understanding of the molecular mechanisms of inflammatory gene regulation by IL-10 and may provide insights necessary to develop strategies for modulating the vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.

ACKNOWLEDGMENTS

Work described in this manuscript was, in part, supported by the American Heart Association Grant 0530350N, to R. K. and National Institute of Health Grants HL63414, HL57516, HL53354, HL66957, and HL60911 to D.W.L. The authors thank Ms. Mickey Neely for secretarial assistance.

Received for publication March 21, 2006. Accepted for publication May 25, 2006.

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