![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Expression by -Irradiation as a Mechanism Contributing to Inflammatory Response in Rat Colon: Modulation by the 5-Aminosalicylic Acid AgonistLaboratory of Radiopathology, Radiological Protection and Human Health Division, Institute for Radioprotection and Nuclear Safety, Fontenay-aux-Roses, France
Received for publication
July 24, 2007
Accepted
December 11, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-irradiation (10 Gy). We tested whether irradiation-induced acute inflammatory response could be modulated pharmacologically with the antiinflammatory properties of 5-aminosalicylic acid (5-ASA) (250 mg/kg/day), which is a PPAR activator. Irradiation drastically reduced mRNA and protein levels of PPAR
and - and of the heterodimer retinoid X receptor (RXR) at 3 days postirradiation. 5-ASA treatment normalized both PPAR and RXR expression at 3 days postirradiation and PPAR at 7 days. By promoting PPAR expression and its nuclear translocation, 5-ASA interfered with the nuclear factor (NF)-
B pathway, both by reducing irradiation-induced NF-B p65 translocation/activation and increasing the expression of nuclear factor-B inhibitor (IB) mRNA and protein. Therefore, 5-ASA prevents irradiation-induced inflammatory processes as well as expression of tumor necrosis factor , monocyte chemotactic protein-1, inducible nitric-oxide synthase, and macrophage infiltration. In addition, 5-ASA restores the interferon /signal transducer and activator of transcription (STAT)-1 and STAT-3 concentrations that were impaired at 3 and 7 days postirradiation and are correlated with suppressor of cytokine signaling-3 repression. Collectively, these results indicate that PPAR agonists may be effective in the prevention of inflammatory processes and immune responses during and after pelvic radiotherapy.
). We previously reported that an inflammatory process induced by abdominal irradiation (Linard et al., 2003) is associated with an imbalance of the Th1/Th2 adaptive immune response that results in repression of genes involved in Th1 differentiation, cytokine responses (IFN) and migratory behavior (Grémy et al., 2006). Differentiation toward the Th2 pathway requires up-regulation of endogenous feedback regulators such as suppressor of cytokine signaling (SOCS)-3 in several inflammatory models (Seki et al., 2003; Li et al., 2006), including abdominal irradiation (Grémy et al., 2006).
One major pathway toward limiting the negative effects of radiotherapy involves reducing inflammation levels and preventing further episodes. Peroxisome proliferator activator receptors (PPARs) belong to the nuclear receptor superfamily of transcription factors, most of which are ligand-dependent transcriptional activators. PPARs dimerize with the retinoid X receptor (RXR), and the binding of PPAR-RXR heterodimers to PPAR response elements (Kliewer et al., 1992) regulates the transcription of target genes. Four PPAR isotypes have thus far been reported and are commonly designated as PPAR, PPAR, PPAR, and PPAR
. PPAR has been found to regulate adipocyte differentiation and metabolism (Oberfield et al., 1999). It is highly expressed in the intestinal and colonic mucosa by both epithelial cells and macrophages but is also present in endothelial cells, vascular smooth muscle cells and colon tumors (Ricote et al., 1998; Sarraf et al., 1999). Some PPAR agonists have been reported to suppress inflammation effectively by inhibiting multiple steps in the nuclear factor (NF)-B signaling pathway (Ricote et al., 1998; Delerive et al., 2001).
PPAR has recently come under scrutiny as a potentially important transcription factor that modulates the inflammatory response of monocytes and macrophages, in particular, by inhibiting the production of nitric oxide (iNOS) and macrophage-derived cytokines, i.e., TNF, IL-1, and IL-6. Evidence also indicates that PPAR is involved at the transcriptional level in the expression of inflammatory mediators; it negatively interferes with the NF-B and AP-1 signaling pathways, independently of DNA binding (Ricote et al., 1998), and this transrepression mechanism is thought to be its primary mode of anti-inflammatory action (Delerive et al., 2001). The potentially beneficial effects of synthetic PPAR ligands are thus being studied in both animal models and humans for their effects on inflammatory diseases, including rheumatoid arthritis, multiple sclerosis (Kawahito et al., 2000), and irritable bowel disease (Dubuquoy et al., 2003). Patients with ulcerative colitis, but not those with Crohn's disease, seem to have reduced levels of PPAR protein in their colonic epithelial lining. Katayama et al. (2003) have suggested similar deficiencies in colitis mouse models, but only in macrophages of the lamina propria. These differences notwithstanding, both studies support the hypothesis that sufficient levels of PPAR are needed to maintain a healthy noninflamed gut. Numerous studies have since reported that various PPAR-activating ligands protect against or delay the onset of colitis. Accordingly, Saubermann et al. (2002) showed that administration of PPAR ligands after dextran sodium sulfate-induced colitis in mice provided little therapeutic benefit but that preventive administration protected against colitis development.
We reasoned that strong PPAR activation would normalize the PPAR-mediated gene response that limits the inflammatory process and might therefore prevent the radiation-induced alteration in immune response. We tested this hypothesis with 5-aminosalicylate (5-ASA), a potent PPAR ligand activator. It inhibits inflammation by scavenging free radicals and thus interfering with the arachidonic acid metabolism (Tromm et al., 1999) but without inducing NF-B activation (Wahl et al., 1998). To our knowledge, no study has reported that irradiation modulates PPAR levels. Our investigation of PPAR expression in a rat abdominal irradiation model reveals that the PPAR gene is extremely sensitive to radiation. Our findings indicate that the PPAR activator can prevent irradiation-induced inflammatory processes in vivo at the molecular level by modulating the NF-B, STAT-1, and STAT-3 pathways as well as by cell infiltration and may thus contribute to restoring a Th1 cytokine profile.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-irradiation (60Co source). 5-ASA (Pentasa; generously provided by Ferring Laboratory, Gentilly, France) (250 mg/kg/day corresponding to 130 mg/kg/day active component) was administered orally once daily for 13 days, starting 7 days before irradiation. Rats were divided into four groups (n = 6): controls, controls treated with 5-ASA, irradiated untreated, and irradiated 5-ASA-treated. At 3 and 7 days after irradiation, animals were sacrificed, and colon tissue was dissected. Entire colon specimens were taken for immunostaining; mucosa layers, separated from the muscularis by scraping, were kept at –80°C for protein extraction and reverse transcription-polymerase chain reaction (RT-PCR). Immunostaining. Colon tissue samples were washed in phosphate-buffered saline (PBS) and then fixed in 40 g/l buffered formaldehyde solution. After dehydration, specimens were embedded in paraffin and sectioned (5 µm).
Macrophage Detection. Sections were dewaxed in xylene, rehydrated by repeated exposure to graded ethanol, and processed to show macrophages. All sections were then immersed in an endogenous peroxidase blocking solution (3% H2O2) and boiled in 10% citrate buffer for antigen retrieval. To reduce nonspecific binding, they were preincubated 10 min at room temperature with the protein blocker (Dako UK Ltd., Ely, Cambridgeshire, UK) and for 1 h with the antibody binding the activated macrophage marker ED1 (catalog number MCA341; 1:50; Serotec, Oxford, UK) and then rinsed in 50 mM Tris-HCl, 0.3 M NaCl, and 0.1% Tween 20. The secondary reagent was the Vector Elite ABC kit (Dako UK Ltd.). Finally, the slides were treated with a NovaRED kit (Vector Laboratories, Burlingame, CA) for color development and then counterstained with Mayer's hemalum.
Preparation of Protein Extracts. Total proteins were obtained by colonic mucosa homogenization in a cold radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO) containing a standard protease inhibitor cocktail. For cytoplasmic and nuclear protein extracts, small aliquots (<0.1 g) were immersed in 1 ml of ice-cold lysis buffer: 10 mM HEPES, pH 7.9, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 1% Igepal, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 5 µl/ml protease inhibitor cocktail (Sigma-Aldrich). They were homogenized on ice with a Dounce homogenizer, kept on ice for 15 min, and then centrifuged at 4°C (12,500 rpm) for 20 s. The supernatant of this cytoplasmic extract was collected into a new tube. The pelleted nuclei were resuspended into 50 to 200 µl of extraction buffer (20 mM HEPES, pH 7.9, 420 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 5 µl/ml protease inhibitor cocktail) and kept on ice for 30 min. The nuclear suspension was centrifuged at 12,500 rpm for 15 min at 4°C to collect the supernatants containing nuclear protein extracts. Protein concentrations of cytoplasmic and nuclear extracts were measured with a modified Bradford method from Bio-Rad S.A (Marnes-la-Coquette, France). The samples were then stored at –80°C.
Immunoblot Analysis. Total, cytosolic and nuclear proteins (25 µg) were boiled in SDS and mercaptoethanol buffer and then separated on a 120 g/l polyacrylamide gel (NuPAGE gels; Invitrogen, Carlsbad, CA) and electroblotted. Polyvinylidene difluoride membranes were incubated with a blocking solution (50 g/l skimmed milk in Tween 20-PBS containing 1 ml/l Tween 20), washed with Tween 20-PBS, and incubated with mouse monoclonal primary antibody directed against PPAR (1/200; Santa Cruz Biochemicals, Santa Cruz, CA), NF-B p65 (1/200; Santa Cruz Biochemicals) and rabbit polyclonal STAT-1 p84/p91 (1/700; Santa Cruz Biochemicals), STAT-3 (1/300; Santa Cruz Biochemicals), and SOCS-3 (1/200; Santa Cruz Biochemicals) for 1 h at room temperature or rabbit polyclonal PPAR (1/200; Santa Cruz Biochemicals), RXR (1/200; Santa Cruz Biochemicals), and IB (1/200; Santa Cruz Biochemicals) overnight at 4°C. After washing, immunodetection was performed with the respective horseradish-linked secondary antibodies (1/1000; Santa Cruz Biochemicals). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin proteins were detected similarly with a rabbit anti-GAPDH polyclonal antibody (1/1000; Santa Cruz Biochemicals) or mouse monoclonal antibody (1/1000; Santa Cruz Biochemicals), respectively, to verify uniformity in gel loading. Chemiluminescence was detected according to the manufacturer's protocol (ECL; Bio-Rad). Mean band densities were quantified with a Las 3000 apparatus (FUJIFILM, Tokyo, Japan) and normalized to the total amount of proteins in the control.
Transcription Factor Activation. NF-Bp65 and PPAR activation were assayed with TransAM NF-B and PPAR kits (Active Motif Inc., Rixensart, Belgium), which include a 96-well plate with an immobilized oligonucleotide containing the NF-B consensus-binding site (5'-GGGACTTTCC-3') for NF-B and a PPAR response element (5'-AACTAGGTCAAAGGTCA-3') for PPAR. The active form of NF-B and PPAR contained in nuclear extract specifically binds to the oligonucleotide, respectively. The primary antibodies that detect NF-B recognize an epitope on p65 that is accessible only when NF-B is activated and bound to its target DNA. The primary antibodies used in the TransAM PPAR kit recognize an accessible epitope on PPAR protein upon DNA binding. A horseradish peroxidase-conjugated secondary antibody was used for the spectrophotometric quantification.
RT-PCR Analysis. The mRNA levels of inflammation-related cytokines and chemokines and of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) were measured by RT-PCR. Total RNA was extracted from the colon mucosa samples with the RNeasy kit (QIAGEN S.A., Courtaboeuf, France). RNA purity and integrity were checked by spectrophotometric analysis and agarose gel electrophoresis. In accordance with the manufacturer's instructions, 1 µg of total RNA was reverse-transcribed into cDNA with random hexamers and the SuperScript II RNase H-(Invitrogen, Cergy-Pontoise, France) in a 20-µl reaction volume. SYBR chemistry (Applied Biosystems, Courtaboeuf, France) was used to amplify PCR in the ABI-Prism 7000 detection system (Applied Biosystems), under the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. The primer sequences, designed with Primer Express software (Applied Biosystems), are listed in Table 1.
|
Result Expression and Statistical Analysis. We used the comparative 
CT method for relative mRNA quantification of target genes, normalized to an endogenous reference (HPRT) and a relevant nonirradiated control, equal to 2–CT. CT is the difference between the mean CT (irradiated sample) and mean CT (nonirradiated sample), where CT is the difference between the mean CT (genes) and the mean CT (HPRT). Each sample was monitored for fluorescent dyes, and signals were regarded as significant if the fluorescence intensity was 10 times higher than the S.D. of baseline fluorescence, defined as threshold cycles (CTs).
All data are expressed as the mean ± S.E.M. for six animals. Data were analyzed by one-way ANOVA followed by a Bonferroni test to determine the significance of the differences.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Rousseaux et al., 2005). PPAR is a negative regulator of macrophage activation (Ricote et al., 1998), and activated macrophages are the major source of TNF, which is both cytotoxic and proinflammatory. We examined the effects of 5-ASA on TNF expression. A previous study in an intestinal inflammatory model found that the oral dosage of controlled-release 5-ASA that was most clinically effective was 100 mg/kg/day (Rousseaux et al., 2005). As Fig. 1A shows, irradiation alone results in multiplies TNF expression at 3 and 7 days postirradiation 7 (p < 0.001) and 2.5 (p < 0.01) times higher, respectively, than in nonirradiated controls. Likewise, the level of MCP-1, known to recruit and activate monocytes and macrophages in tissue, tripled (p < 0.01) at 3 days (Fig. 1B). In addition, because activated macrophages may be one source of iINOS induction, we analyzed iNOS expression and found that its mRNA was 7 times higher than in controls at 3 days postirradiation and 3 times higher at 7 days (Fig. 1C). Immunostaining of macrophages confirmed that overexpression of TNF, MCP-1 and iNOS was correlated with elevated levels of ED1-positive macrophages in the lamina propria at 3 and 7 days postirradiation (Fig. 1D). 5-ASA pretreatment markedly decreased (p < 0.05) the irradiation-induced overexpression of TNF, MCP-1, and iNOS at 3 days postirradiation and resulted in normalized (p < 0.05 versus irradiated nontreated controls) TNF and iNOS expression at 7 days. These results were consistent with the histological anti-inflammatory benefits of 5-ASA, seen in the drastically reduced macrophage infiltration in the colonic mucosa.
|
Effect of 5-ASA on Irradiation-Induced Alterations in the Expression of PPAR-Related Genes. To assess the effects of increasing PPAR levels in vivo during the irradiation protocol, we determined these levels after irradiation and treatment (and corrected for the mRNA levels of HPRT housekeeping gene). In nontreated animals, 10-Gy abdominal irradiation induced a significant, even drastic, decrease in PPAR (–50%; p < 0.05) and PPAR (–60%; p < 0.01) (Fig. 2, A and B) levels, at 3 days postirradiation compared with nonirradiated controls. These reductions drove repression (–50%, p < 0.01) of RXR heterodimers (Fig. 2C). Western blot analysis of total proteins confirmed the irradiation-induced reduction in PPAR and RXR expression at 3 days, indicating that the reduction in PPAR protein levels is associated with the reduction in mRNA levels. Pretreatment with 5-ASA limited repression of both PPAR and RXR at 3 days and contributed to the greater expression, compared with controls, of both PPAR and RXR (both mRNA and protein) at 7 days postirradiation.
|
Activation of PPARs results in a cascade of reactions, including nuclear translocation (Delerive et al., 2001; Rousseaux et al., 2005). Western blot analysis of the cytosol and nucleus (Fig. 3, A–C) thus extended and confirmed the finding that the decreased protein levels led to decreased nuclear PPAR and - and RXR at day 3. By up-regulating mRNA and total protein, 5-ASA treatment normalized their translocation levels. In addition, analysis of PPAR activation (Fig. 3D) showed that irradiation inhibited by approximately 2-fold the PPAR DNA-binding capacity at 3 and 7 days. This activation capacity was significantly amplified by 5-ASA treatment at 7 days postirradiation.
|
5-ASA Attenuated Irradiation-Related NF-B Translocation. Previous studies suggested that PPARs modulate inflammation through their interaction with the NF-B signaling pathway, either by reducing NF-B DNA binding to target genes or by inducing the expression of IB (Delerive et al., 2000). We therefore used Western blotting of nuclear and cytosolic extracts in the colon to determine whether the anti-inflammatory effects of 5-ASA resulted from interference with NF-B p65 nuclear translocation after irradiation (Fig. 4A). The results showed that p65 protein levels in the nucleus doubled after irradiation alone at 3 and 7 days (p < 0.01) without any modification of the NF-B p65 protein level in the cytosol. Nonetheless, irradiated animals with 5-ASA treatment had normal nuclear NF-B P65 protein levels (p < 0.05 versus irradiated groups) at day 7. NF-B dimers, after translocation into the nucleus, activate appropriate target genes. In control rats, a basal NF-B p65 DNA-binding activity was found in the nuclear extracts of the mucosa layer; it disappeared in the presence of an excess amount of soluble oligonucleotide containing a wild-type NF-B consensus-binding site (data not shown). This finding confirms the specificity of the p65 DNA-binding activity observed. At 3 and 7 days postirradiation, the activity in the nuclear extract was significantly 2-fold higher than in the control (p < 0.001) (Fig. 4B). 5-ASA treatment reduced significantly the p65 DNA-binding activity. These data demonstrated that the total amount of translocated p65 protein by irradiation was activated, and 5-ASA interferes with irradiation-induced increase of p65 DNA-binding activity.
|
B requires binding to IB inhibitor proteins. Irradiation induced a significant decrease in IB expression, confirmed for both mRNA (–30%; p < 0.05) and protein (–70%; p < 0.05) in total protein extract at 3 days postirradiation. 5-ASA pretreatment restored the IB mRNA level at day 3 and was associated with an IB protein level at day 7 that was 5 times higher (p < 0.05) than in the irradiated-only rats. This finding indicates that 5-ASA maintains the NF-B p65 protein in cytosol by regulating IB expression (Fig. 4C).
5-ASA Effect on IFN/STAT-1 Expression. PPARs are known to affect immune response by regulating the Th1/Th2 balance (Mueller et al., 2003). As previously reported (Grémy et al., 2006), abdominal irradiation leads to decreased Th1 cytokine IFN expression. Figure 5A confirms the drastic repression of irradiation-induced IFN (–75 and 60%, p < 0.01, at days 3 and 7, respectively). 5-ASA treatment normalized the IFN expression (p < 0.05 versus irradiated rats) at day 7. Because IFN signaling serves mainly to activate STAT-1, we sought to determine whether IFN repression in irradiated mucosa was correlated with STAT-1 repression. Western blot analysis showed that STAT-1 immunoreactivity in nuclear and cytosolic extracts was significantly lower at day 3 than in controls (Fig. 5B). At day 7, STAT-1 protein levels in nuclear extracts remained low (p < 0.05), but in the animals pretreated with 5-ASA treatment, they returned to normal then (p < 0.05 versus irradiated).
|
Expression of the SOCS-3. We focused our attention on how irradiation might inhibit IFN/STAT-1 expression. We considered the possibility that endogenous feedback regulators of cytokine activities, such as SOCS-1 and SOCS-3, might be involved in these events because they have been identified as negative regulators of the IFN/STAT-1 pathway (Schreiber et al., 2002). We observed no modification of SOCS-1 expression postirradiation (Fig. 6A). However, SOCS-3 expression increased markedly at 3 and 7 days postirradiation (by multiples of 13 and 3, respectively, p < 0.01 and p < 0.001). Western blot analysis on day 3 confirmed the elevation in the SOCS-3 protein level (Fig. 6B). 5-ASA pretreatment resulted in normal SOCS-3 mRNA and protein level expressions at day 3 postirradiation.
|
). We observed a significant reduction in STAT-3 immunoreactivity in nuclear and cytosolic extracts at day 3 compared with controls (Fig. 6C), as well as repression of radiation-induced STAT-1 protein expression. 5-ASA treatment counterbalanced the irradiation-induced STAT-3 repression by elevating STAT-3 protein levels in both cytosol and nucleus at day 3. This STAT-3 elevation remained significant in the cytosol at 7 days postirradiation. These data support the hypothesis that PPAR ligands establish STAT-1 and STAT-3 levels by regulating the SOCS-3 gene. |
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
agonist, 5-ASA, exerts potent anti-inflammatory effects in the rat colon after abdominal irradiation. 5-ASA therapy has been widely used in patients with inflammatory bowel disease (van Bodegraven and Mulder, 2006), and PPAR, the key receptor for 5-ASA, mediates its main effects in the colon (Rousseaux et al., 2005). In addition to this therapeutic effect of prophylactically administered 5-ASA, some reports show that in experimentally induced colitis, PPAR ligands consistently reduce lesion despite destruction of the epithelial cell layer (Desreumaux et al., 2001). The utility of 5-ASA drugs in preventing acute enteritis during radiotherapy has been a much debated subject (Rubenstein et al., 2004). The benefic effect of 5-ASA drugs remained controversial because of the different mechanisms involved in this severe radiation complication.
In animal models, irradiation-induced intestinal inflammatory processes have previously been characterized by increased cytokine production, including those involved in Th2-mediated-immune polarization (Grémy et al., 2006). PPAR ligands have been shown to reduce cytokine expression in activated macrophages (Ricote et al., 1998), an effect that appears to be independent of PPAR expression (Welch et al., 2003). The main cellular sources of PPAR in colonic mucosa are epithelial cells and macrophages (Jain et al., 1998). Previous studies have suggested that inflammation may affect PPAR expression. In clinical settings, PPAR expression in colonic epithelial cells is impaired in patients with ulcerative colitis but remains normal in inflammatory cells (Dubuquoy et al., 2003). Our analysis of PPAR expression revealed its dramatic impairment at 3 days postirradiation, related, moreover, to repression of both PPAR and the RXR heterodimers. Here, however, unlike in ulcerative colitis, the PPAR defect is not due to a general alteration in nuclear factor expression. These findings suggest that reduced PPAR expression may contribute to the overwhelming colonic inflammation associated with abdominal irradiation. This acute gastrointestinal inflammation has only recently been recognized as the source of the gastrointestinal symptoms that affect quality of life and may even lead to stopping radiotherapy treatment (Andreyev, 2005).
Of the different hypotheses for the diminution in PPAR levels during inflammation, irradiation-induced alteration of epithelial cells seems particularly likely and may explain PPAR repression, although no histological differences were seen after 5-ASA treatment. Despite alterations of the epithelial cell layer, however, PPAR ligands consistently reduce lesion intensity in several colitis models (Katayama et al., 2003; Rousseaux el al, 2005). Additional mechanisms may also play a role after irradiation. For example, nitration of PPAR proteins inhibits the ability of the nuclear receptor to translocate, and the peroxynitrite produced by superoxide and NO radicals may cause this nitration, either directly by the irradiation-induced production of ROS species or indirectly by overexpression of TNF and iNOS. In a recent study, some PPAR ligands have proved effective against nitration related to rheumatoid arthritis and TNF-stimulated macrophage cell line (Shiojiri et al., 2002). We hypothesize from our findings that the suppression of irradiation-induced iNOS overexpression by 5-ASA may have contributed to decreasing PPAR nitration and resulted in restoring PPAR levels, both total protein and in the nucleus.
At this time, nothing at all is known about regulation of PPAR expression. However, evidence suggests that 5-ASA is able to bind PPAR, activate it, enhance its expression, and promote its translocation from the cytoplasm to the nucleus, perhaps in a positive-feedback loop (Rousseaux et al., 2005). This point is particularly important in our irradiation model where the protein PPAR normalization induced by 5-ASA was also associated with increased translocation and a delayed (day 7) activation. It is interesting to note that 5-ASA normalizes both PPAR and RXR. In reality, the PPAR activation required a cotranslocation with the heterodimer RXR (Kliewer et al., 1992) only restored at day 7 by 5-ASA. These results indicated that the 5-ASA beneficial effect acted by inducing PPARs/RXR coexpression. Although the therapeutic efficacy of 5-ASA has been recognized in diverse clinical situations (van Bodegraven and Mulder, 2006), this agent was originally developed without knowledge of its molecular target. Our data may define its efficacy as the dual activation of PPAR and PPAR.
In vivo studies have shown that PPAR ligands suppress inflammatory response by attenuating cytokine/chemokine production in mouse models of colitis (Desreumaux et al., 2001; Saubermann et al., 2002; Katayama et al., 2003). 5-ASA may thus have widespread beneficial effects in our irradiation model, where the reduction of macrophages resulted in decreased levels of such inflammatory mediators as TNF, MCP-1, and iNOS. The beneficial effect of 5-ASA on inflammatory mediators in our study occurred at 3 days postirradiation, a point at which PPAR and - are expressed only weakly. These data support the hypothesis by Crosby et al. (2005) from their study of PPAR-deficient macrophages that PPAR is not necessary for the successful inhibition of inflammatory mediators, in particular, iNOS, by synthetic agonists.
PPAR activator-repressed inflammatory genes inhibit signaling of NF-B transcriptional activity. Proposed mechanisms include PPAR interaction with the p65/p50 subunit and thus the inhibition of IB protein degradation (Delerive et al., 2000). In this study, the interaction between PPARs and NF-B has been restricted to the NF-B p65 subunit, where an activation of the total amount of p65 translocation induced by irradiation has been observed. 5-ASA significantly reduced irradiation-induced NF-B nuclear translocation/activation. Inhibition of p65 translocation by 5-ASA was only observed at day 7. Kennedy et al. (1999) demonstrated that 5-ASA had no effect on the NF-B nuclear translocation. In fact, a timing issue may have occurred for this discrepancy. The therapeutic value of 5-ASA require a PPAR translocation into nucleus (seen at day 3 in this study), a recruitment of coactivator (Rousseaux et al., 2005), and a IB expression that occurred at day 3 in this study and its stabilization in cytoplasm (Delerive et al., 2000); the NF-B translocation alteration was the issue of these different steps. In addition, this delayed response is consistent with the hypothesis that PPAR-mediated inhibition of NF-B-driven gene transcription increases with exposure to PPAR ligands (Delerive et al., 1999) and thus suggests a complementary mechanism. NF-B activity is tightly controlled by the degradation of IB, which sequesters inactive NF-B dimers in cytosol. It is interesting to note that PPAR ligands induced IB mRNA and protein expression (Delerive et al., 2000) when PPAR ligands inhibited IB-kinase complex activity, thereby both preventing IB degradation and increasing IB nuclear traffic (Castrillo et al., 2000), without increasing the protein level, as far as we know. Our data show that the marked decrease in IB mRNA and protein elicited by irradiation was substantially impaired by 5-ASA, and the protein overexpression at 7 days confirms that impairment of 5-ASA may thus act like dual PPAR and - ligands on NF-B signaling by overexpressing IB. A discrepancy was observed between the magnitude of IB level (5-fold) and the only 2-fold decrease of p65 activity at day 7. PPAR inhibits NF-B-driven transcription by physically interacting with both p65 and p50 (Delerive et al., 1999); this study was only restricted to p65 activity.
Previous reports noted that STAT signaling may be involved in the anti-inflammatory action of PPAR ligands (Ricote et al., 1998). In this study, we showed that 5-ASA can act at two points: by restoring or increasing the irradiation-induced modulation of STAT-1 correlated with repression of SOCS-3, which is the regulator of cytokine signaling involved in the STAT pathway. SOCS-1 probably inhibits IFN signaling most strongly, and it acts by suppressing STAT-1 phosphorylation (Brysha et al., 2001). SOCS-3 plays an essential role as a negative inhibitor of IL-6 by interfering with STAT-3 (Ihle, 2001). However, macrophage activation models indicate that SOCS-3 overexpression may also inhibit IFN/STAT-1 signaling (Stoiber et al., 2001; Ekchariyawat et al., 2005). Our study thus extends previous reports (Han et al., 2002; Grémy et al., 2006) that irradiation reduces IFN levels associated with STAT-1 signaling. It is tempting to conclude that irradiation-induced SOCS-3 can interfere with IFN/STAT-1 signaling and STAT-3. Classic PPAR agonists rapidly induce SOCS-1 and SOCS-3 transcription, thus suppressing STAT phosphorylation and in turn repressing IFN (Chen et al., 2003; Park et al., 2003). Our data are not wholly consistent with these reports but indicate instead that the PPAR ligand 5-ASA restores IFN/STAT-1 and STAT-3 levels that were altered by irradiation. Based on observations that immune Th2 differentiation is enhanced in SOCS-3-transgenic mice, whereas expression of dominant-negative SOCS-3 interferes with Th2 differentiation (Li et al., 2006) and that irradiation shifts the immune response to Th2 cell polarization (Grémy et al., 2006), we can hypothesize that SOCS-3 induction would have a dramatic effect on the immune system. SOCS-3 repression by 5-ASA may be a key element in minimizing the inflammatory and immune effects of irradiation. Weber et al. (2003) show that PPAR agonists initiate T cell immune response, especially Th1 response, by reducing IFN/STAT-1 signaling. Confirming the findings of Mueller et al., we demonstrated that the PPAR ligand may also regulate induction of a Th2 type immune response notably after irradiation, and we think that the effects of PPAR ligation in immune response are highly pleomorphic.
|
Footnotes |
|---|
ABBREVIATIONS: IFN, interferon; SOCS, suppressor of cytokine signaling; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid receptor; NF, nuclear factor; IB, nuclear factor-B inhibitor; iNOS, inducible nitric-oxide synthase; TNF, tumor necrosis factor; IL, interleukin; 5-ASA, 5-aminosalicylate; STAT, signal transducer and activator of transcription; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; HPRT, hypoxanthine-guanine phosphoribosyltransferase; CT, threshold cycle; MCP, monocyte chemotactic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Address correspondence to: Dr. Christine Linard, Institut de Radioprotection et de Sûreté Nucléaire, Direction de la Radioprotection de l'Homme, Laboratoire de Radiopathology, B.P. no. 17, F-92262 Fontenay-aux-Roses Cedex, France. E-mail: christine.linard{at}irsn.fr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Andreyev J (2005) Gastrointestinal complications of pelvic radiotherapy: are they of any importance? Gut 54: 1051–1054.
Brysha M, Zhang JG, Bertolino P, Corbin JE, Alexander WS, Nicola NA, Hilton DJ, and Starr R (2001) Suppressor of cytokine signaling-1 attenuates the duration of interferon signal transduction in vitro and in vivo. J Biol Chem 276: 22086–22089.
Castrillo A, Diaz-Guerra MJ, Hortelano S, Martin-Sanz P, and Bosca L (2000) Inhibition of IB kinase and IB phosphorylation by 15-deoxy-delta12,14-prostaglandin J2 in activated murine macrophages. Mol Cell Biol 20: 1692–1698.
Chen CW, Chang YH, Tsi CJ, and Lin WW (2003) Inhibition of IFN--mediated inducible nitric oxide synthase induction by the peroxisome proliferator-activated receptor agonist, 15-deoxy-delta12,14-prostaglandin J2, involves inhibition of the upstream Janus kinase/STAT1 signaling pathway. J Immunol 171: 979–988.
Crosby MB, Svenson JL, Zhang J, Nicol CJ, Gonzalez FJ, and Gilkeson GS (2005) Peroxisome proliferation-activated receptor (PPAR) is not necessary for synthetic PPAR agonist inhibition of inducible nitric-oxide synthase and nitric oxide. J Pharmacol Exp Ther 312: 69–76.
Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G, and Staels B (1999) Peroxisome proliferator-activated receptor negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-B and AP-1. J Biol Chem 274: 32048–32054.
Delerive P, Fruchart JC, and Staels B (2001) Peroxisome proliferator-activated receptors in inflammation control. J Endocrinol 169: 453–459.[Abstract]
Delerive P, Gervois P, Fruchart JC, and Staels B (2000) Induction of IB expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor- activators. J Biol Chem 275: 36703–36707.
Desreumaux P, Dubuquoy L, Nutten S, Peuchmaur M, Englaro W, Schoonjans K, Derijard B, Desvergne B, Wahli W, Chambon P, et al. (2001) Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor (PPAR ) heterodimer: a basis for new therapeutic strategies. J Exp Med 193: 827–838.
Dubuquoy L, Jansson EA, Deeb S, Rakotobe S, Karoui M, Colombel JF, Auwerx J, Pettersson S, and Desreumaux P (2003) Impaired expression of peroxisome proliferator-activated receptor in ulcerative colitis. Gastroenterology 124: 1265–1276.[CrossRef][Medline]
Ekchariyawat P, Pudla S, Limposuwan K, Arjcharoen S, Sirisinha S, and Utaisincharoen P (2005) Burkholderia pseudomallei-induced expression of suppressor of cytokine signaling 3 and cytokine-inducible src homology 2-containing protein in mouse macrophages: a possible mechanism for suppression of the response to interferon stimulation. Infect Immun 73: 7332–7339.
Frieri G, Giacomelli R, Pimpo M, Palumbo G, Passacantando A, Pantaleoni G, and Caprilli R (2000) Mucosal 5-aminosalicylic acid concentration inversely correlates with severity of colonic inflammation in patients with ulcerative colitis. Gut 47: 410–414.
Grémy O, Benderitter M, and Linard C (2006) Caffeic acid phenethyl ester modifies the Th1/Th2 balance in ileal mucosa after -irradiation in the rat by modulating the cytokine pattern. World J Gastroenterol 12: 4996–5004.[Medline]
Han SK, Song JY, Yun YS, and Yi SY (2002) Gamma irradiation-reduced IFN- expression, STAT1 signals, and cell-mediated immunity. J Biochem Mol Biol 35: 583–589.[Medline]
Ihle JN (2001) The Stat family in cytokine signaling. Curr Opin Cell Biol 13: 211–217.[CrossRef][Medline]
Jain S, Pulikuri S, Zhu Y, Qi C, Kanwar YS, Yeldandi AV, Rao MS, and Reddy JK (1998) Differential expression of the peroxisome proliferator-activated receptor (PPAR) and its coactivators steroid receptor coactivator-1 and PPAR-binding protein PBP in the brown fat, urinary bladder, colon, and breast of the mouse. Am J Pathol 153: 349–354.
Katayama K, Wada K, Nakajima A, Mizuguchi H, Hayakawa T, Nakagawa S, Kadowaki T, Nagai R, Kamisaki Y, Blumberg RS, et al. (2003) A novel PPAR gamma gene therapy to control inflammation associated with inflammatory bowel disease in a murine model. Gastroenterology 124: 1315–1324.[CrossRef][Medline]
Kawahito Y, Kondo M, Tsubouchi Y, Hashiramoto A, Bishop-Bailey D, Inoue K, Kohno M, Yamada R, Hla T, and Sano H (2000) 15-deoxy-delta12,14-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest 106: 189–197.[Medline]
Kennedy M, Wilson L, Szabo C, and Salzman AL (1999) 5-aminosalicylic acid inhibits iNOS transcription in human intestinal epithelial cells. Int J Mol Med 4: 437–443.[Medline]
Kliewer SA, Umesono K, Noonan DJ, Heyman RA, and Evans RM (1992) Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358: 771–774.[CrossRef][Medline]
Li Y, Chu N, Rostami A, and Zhang GX (2006) Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo. J Immunol 177: 1679–1688.
Linard C, Ropenga A, Vozenin-Brotons MC, Chapel A, and Mathe D (2003) Abdominal irradiation increases inflammatory cytokine expression and activates NF-B in rat ileal muscularis layer. Am J Physiol 285: G556–G565.
Mueller C, Weaver V, Vanden Heuvel JP, August A, and Cantorna MT (2003) Peroxisome proliferator-activated receptor ligands attenuate immunological symptoms of experimental allergic asthma. Arch Biochem Biophys 418: 186–196.[CrossRef][Medline]
Naka T, Fujimoto M, and Kishimoto T (1999) Negative regulation of cytokine signaling: STAT-induced STAT inhibitor. Trends Biochem Sci 24: 394–398.[CrossRef][Medline]
Oberfield JL, Collins JL, Holmes CP, Goreham DM, Cooper JP, Cobb JE, Lenhard JM, Hull-Ryde EA, Mohr CP, Blanchard SG, et al. (1999) A peroxisome proliferator-activated receptor ligand inhibits adipocyte differentiation. Proc Natl Acad Sci U S A 96: 6102–6106.
Park EJ, Park SY, Joe EH, and Jou I (2003) 15d-PGJ2 and rosiglitazone suppress Janus kinase-STAT inflammatory signaling through induction of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 in glia. J Biol Chem 278: 14747–14752.
Ricote M, Li AC, Willson TM, Kelly CJ, and Glass CK. (1998) The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391: 79–82.[CrossRef][Medline]
Rousseaux C, Lefebvre B, Dubuquoy L, Lefebvre P, Romano O, Auwerx J, Metzger D, Wahli W, Desvergne B, Naccari GC, et al. (2005) Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-. J Exp Med 201: 1205–1215.
Rubenstein EB, Peterson DE, Schubert M, Keefe D, McGuire D, Epstein J, Elting LS, Fox PC, Cooksley C, and Sonis ST (2004) Clinical practice guidelines for the prevention and treatment of cancer therapy-induced oral and gastrointestinal mucositis. Cancer 100 (Suppl 9): 2026–2046.[CrossRef][Medline]
Sarraf P, Mueller E, Smith WM, Wright HM, Kum JB, Aaltonen LA, de la Chapelle A, Spiegelman BM, and Eng C (1999) Loss-of-function mutations in PPAR associated with human colon cancer. Mol Cell 3: 799–804.[CrossRef][Medline]
Saubermann LJ, Nakajima A, Wada K, Zhao S, Terauchi Y, Kadowaki T, Aburatani H, Matsuhashi N, Nagai R, and Blumberg RS (2002) Peroxisome proliferator-activated receptor agonist ligands stimulate a Th2 cytokine response and prevent acute colitis. Inflamm Bowel Dis 8: 330–339.[CrossRef][Medline]
Schreiber S, Rosenstiel P, Hampe J, Nikolaus S, Groessner B, Schottelius A, Kuhbacher T, Hamling J, Folsch UR, and Seegert D (2002) Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease. Gut 51: 379–385.
Seki Y, Inoue H, Nagata N, Hayashi K, Fukuyama S, Matsumoto K, Komine O, Hamano S, Himeno K, Inagaki-Ohara K, et al. (2003) SOCS-3 regulates onset and maintenance of TH2-mediated allergic responses. Nat Med 9: 1047–1054.[CrossRef][Medline]
Shiojiri T, Wada K, Nakajima A, Katayama K, Shibuya A, Kudo C, Kadowaki T, Mayumi T, Yura Y, and Kamisaki Y (2002) PPAR ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice. Eur J Pharmacol 448: 231–238.[CrossRef][Medline]
Stoiber D, Stockinger S, Steinlein P, Kovarik J, and Decker T (2001) Listeria monocytogenes modulates macrophage cytokine responses through STAT serine phosphorylation and the induction of suppressor of cytokine signaling 3. J Immunol 166: 466–472.
Tromm A, Griga T, and May B (1999) Oral mesalazine for the treatment of Crohn's disease: clinical efficacy with respect to pharmacokinetic properties. Hepatogastroenterology 46: 3124–3135.[Medline]
van Bodegraven AA and Mulder CJ (2006) Indications for 5-aminosalicylate in inflammatory bowel disease: is the body of evidence complete? World J Gastroenterol 12: 6115–6123.[Medline]
Wahl C, Liptay S, Adler G, and Schmid RM (1998) Sulfasalazine: a potent and specific inhibitor of nuclear factor B. J Clin Invest 101: 1163–1174.[Medline]
Weber SM, Scarim AL, and Corbett JA (2003) Inhibition of IFN--induced STAT1 activation by 15-deoxy-delta12,14-prostaglandin J2. Am J Physiol Endocrinol Metab 284: E883–E891.
Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, and Glass CK (2003) PPAR and PPAR negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci U S A 100: 6712–6717.
This article has been cited by other articles:
|
|
 |
|
  O. De Backer, E. Elinck, E. Priem, L. Leybaert, and R. A. Lefebvre Peroxisome Proliferator-Activated Receptor {gamma} Activation Alleviates Postoperative Ileus in Mice by Inhibition of Egr-1 Expression and Its Downstream Target Genes J. Pharmacol. Exp. Ther., November 1, 2009; 331(2): 496 - 503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R Marion-Letellier, P Dechelotte, M Iacucci, and S Ghosh Dietary modulation of peroxisome proliferator-activated receptor gamma Gut, April 1, 2009; 58(4): 586 - 593. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) and in 5-ASA-treated (
). Real-time PCRs of TNF
