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

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

SPECIFIC AIMS

Mononuclear phagocytes are likely participants in the host response to vascular injury, via the secretion of cytokines and chemokines. tumor necrosis factor (TNF)-alpha (hereafter referred as TNF), produced largely by activated monocytes/macrophages, is known to be negatively associated with restenosis and atherosclerosis. Thus regulation of TNF production via deactivation of inflammatory cells within revascularized arteries could represent a desirable approach for restenosis prevention. Interleukin (IL)-10 (IL-10), a potent antiinflammatory cytokine is a strong deactivator of monocytes and suppressor of the synthesis of TNF and other proinflammatory cytokines. However, the therapeutic effects of IL-10 treatment on the inhibition of injury-induced arterial remodeling and the molecular signaling that governs IL-10 inhibition of inflammatory gene expression are poorly understood. In the present study, we tested the hypothesis that IL-10 treatment inhibits injury-induced monocyte accumulation in injured mouse carotid arteries principally by suppressing TNF expression by enhancing post-transcriptional degradation of TNF mRNA.

PRINCIPAL FINDINGS:

1. Systemic recombinant IL-10 inhibits in vivo inflammatory cell infiltration and TNF expression in denuded mouse carotid arteries
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. Arterial cross sections (day 21 postinjury) were stained with macrophage/monocyte specific antibody (Ab), M4/80, and counted in six different fields/artery by two blinded investigators. As shown in representative Fig. 1 A and quantified in Fig. 1B , IL-10 treatment significantly reduced the number of infiltrating inflammatory cells. Reduction in infiltrating monocytes/macrophages in IL-10 treated mice arteries was strongly correlated with the largely abrogated TNF immunostaining (Fig. 1C ).

View larger version (47K): [in this window] [in a new window]   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.

2. IL-10 suppresses LPS-induced TNF expression accelerating TNF mRNA destabilization

3. AU-rich elements in TNF3' untranslated region are necessary to confer IL-10 sensitivity and mRNA instability to otherwise stable chloroamphenicol acetyltransferase reporter mRNA

4. IL-10 inhibits HuR expression and binding to TNF-3' untranslated region ARE sequences
Cytoplasmic extracts from variously treated U937 cells were allowed to react with a radiolabeled ([-³²P]-uridine triphosphate) single-stranded RNA fragment corresponding to the sense orientation of the 3' untranslated region (UTR)-ARE of the human TNF mRNA prepared by in vitro transcription (IVT) in RNA-EMSA experiments (Fig. 2A). TNF3'UTR-ARE formed three 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. 2A ). Both of these complexes represented specific binding activities to TNF-ARE since 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 since disruption of overlapping UA pentamers (TNF3'UTR-AREmut) 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 prior to the addition of labeled probe "supershifted" complex II from LPS-treated extracts without affecting complexes I and III (Fig. 2B ). 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. 2B ). These data implied that IL-10 might modulate the expression of proteins that 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 Figure 2C , HuR expression was substantially reduced by IL-10, whereas TTP expression had no effect, suggesting that TNF mRNA destabilization by IL-10 may partially result from IL-10-mediated inhibition of HuR.

View larger version (15K): [in this window] [in a new window]   Figure 2. 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 ([-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.

5. HuR silencing by siRNA abrogates IL-10 effects on enhanced TNF mRNA degradation

6. IL-10-inhibits LPS-induced p38MAPK activation and this modulation of p38MAPK partly mediate IL-10-mediated TNF mRNA destabilization

CONCLUSIONS AND SIGNIFICANCE

Experimental and clinical data indicate that inflammation is of major importance to the restenotic process. Monocytes have been hypothesized to serve as markers, initiators, and promoters of arterial occlusive disease. Therefore, control of postinjury inflammatory response represents a desirable and significant approach to curb restenosis. Information regarding the antiinflammatory effects of IL-10 to the neointimal thickening and restenosis following arterial injury is limited and has only begun to emerge lately. Moreover, not many previous studies have looked either at the specific inflammatory gene repression in response to IL-10 or the mechanisms of such inhibition, thus necessitating further mechanistic studies and signaling studies. Our data presented in this study thus not only demonstrate physiological in vivo effect of IL-10 therapy on monocyte accumulation and TNF expression in arterial injury model but also details the mechanistic insights into IL-10 suppressive action using TNF as a model gene.

Over the years, it has become apparent that AREs can mediate destabilization, as well as stabilization of certain mRNAs largely in a stimulus/gene-specific manner, depending on the modulations in transfactors/proteins that bind to AREs. Certain ARE-binding proteins purported to modulate TNF regulations have been described. These include, TIA, TIAR, TTP, and HuR. Whether IL-10 modulates the binding kinetics, expression or function of these TNF-ARE binding proteins is, however, not elucidated. Our data thus provide a novel finding that IL-10 treatment inhibits the expression of TNF mRNA stabilizing protein HuR and represses the binding of HuR to TNF ARE resulting in the enhanced degradation of TNF mRNA.

The signaling mechanisms involved in IL-10-mediated TNF mRNA decay are poorly understood. Our data, therefore, attempts 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. 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. 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.

The detailed mechanisms by which IL-10 targets p38 signaling are not yet known. One possibility could be that IL-10-induced STAT3 activation, in turn, activates factor(s) capable of interfering with p38 signaling. These assertions, however, remain to be tested and confirmed in our experimental system. Possible interactions of IL-10R signaling to modules affecting TNF gene expression are shown in Fig. 3 . 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.

View larger version (31K): [in this window] [in a new window]   Figure 3. Possible interactions of IL-10R signaling to modules affecting TNF gene expression. LPS binding/receptor complex leads to the transmission of p38MAPK signals activating TNF mRNA stability/ translation (open circles and arrows). At similar or consecutive time points, it leads to the activation of stability and translation determining factors that act in a negative fashion, apparently via p38-independent mechanisms (gray shaded circles and arrows). On the other hand, IL-10 receptor engagement may transmit signals toward SOCS3 activation, building up a negative regime targeting p38/MK2 activating signals toward ARE-dependent TNF mRNA stability/translation, likely mediated by the selective binding of various transfactors such as TTP (translation) and HuR (mRNA stability).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6084fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6084fjev1 20/12/2112    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rajasingh, J. Articles by Kishore, R. Search for Related Content PubMed PubMed Citation Articles by Rajasingh, J. Articles by Kishore, R.