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the Institut Pasteur de Lille (R.P., C.B., O. Briand, O. Barbier, C.D., F.P., J.-C.F., C.G., B.S.), Deepartement d’Atheeroscleerose
INSERM, U545 (R.P., C.B., O. Briand, O. Barbier, C.D., F.P., J.-C.F., C.G., B.S.)
Universitee de Lille 2 (R.P., C.B., O. Briand, O. Barbier, C.D., F.P., J.-C.F., C.G., B.S.)
INSERM, U547 (G.W., D.D.), Institut Pasteur de Lille, Lille, France.
Abstract
Statins are inhibitors of 3-hydroxy-3-methylglutaryleCcoenzyme A (HMG-CoA) reductase used in the prevention of cardiovascular disease (CVD). In addition to their cholesterol-lowering activities, statins exert pleiotropic antiinflammatory effects, which might contribute to their beneficial effects not only on CVD but also on lipid-unrelated immune and inflammatory diseases, such as rheumatoid arthritis, asthma, stroke, and transplant rejection. However, the molecular mechanisms involved in these antiinflammatory properties of statins are unresolved. Here we show that the peroxisome proliferatoreCactivated receptor (PPAR) mediates antiinflammatory effects of simvastatin in vivo in models of acute inflammation. The inhibitory effects of statins on lipopolysaccharide-induced inflammatory response genes were abolished in PPAR-deficient macrophages and neutrophils. Moreover, simvastatin inhibited PPAR phosphorylation by lipopolysaccharide-activated protein kinase C (PKC) . A constitutive active form of PKC inhibited nuclear factor B transrepression by PPAR whereas simvastatin enhanced transrepression activity of wild-type PPAR, but not of PPAR mutated in its PKC phosphorylation sites. These data indicate that the acute antiinflammatory effect of simvastatin occurs via PPAR by a mechanism involving inhibition of PKC inactivation of PPAR transrepression activity.
Key Words: inflammation macrophages neutrophils nuclear receptors statins PKC
Introduction
Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryleCcoenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, are widely prescribed for the treatment of hypercholesterolemia.1 In addition to plasma lipid-modulating action, statins exert pleiotropic antiinflammatory effects, which might contribute to their beneficial effects on cardiovascular disease (CVD).2 Emerging evidences also suggest beneficial therapeutic activities of statins in immune and inflammatory diseases such as multiple sclerosis, Alzheimer’s disease, ischemic stroke, transplant rejection, rheumatoid arthritis, and asthma.3eC6 Several clinical observations indicate that these effects cannot be attributed to their cholesterol-lowering activities only.7 Statin therapy decreases plasma concentrations of inflammatory markers, such as C-reactive protein (CRP), within 1 week after treatment initiation, before any lipid changes are observed.8 Statin treatment reduces the incidence of ischemic stroke for which plasma cholesterol levels are not considered a risk factor.9 Moreover, statins also exert antiinflammatory actions in animal models, which are resistant to their hypolipidemic actions.10 In models of acute and chronic inflammation, statins inhibit endothelial adhesion and transendothelial migration of leukocytes to sites of inflammation,10 acting both on endothelial cells and leukocytes. Statins modulate macrophage functions by inhibiting the activation of inflammatory response genes, such as interleukin (IL)-1b and IL-6, tumor necrosis factor (TNF) , metalloproteinase (MMP)-2, and MMP-9, and inducible nitric oxide synthase (iNOS).11 These antiinflammatory actions of statins are attributed to their ability to modulate signal transduction pathways activating proinflammatory transcription factors, such as nuclear factor (NF) B.12
PPAR is a nuclear receptor that regulates gene expression by binding with its heterodimeric partner the retinoid-X-receptor (RXR) to PPAR-responsive elements (PPREs). PPAR not only regulates lipid metabolism13 but also exerts pronounced antiinflammatory activities.14 Clinical trials have shown that fibrates decrease inflammation and have beneficial effects on CVD and stroke.14,15 In animals, PPAR deficiency induces a prolonged inflammatory response in a mouse ear-swelling model. PPAR exerts antiinflammatory activities by negatively interfering with proinflammatory signaling pathways including NFB. This molecular action is exemplified by the inhibition of inflammatory induction of genes, such as vascular cell adhesion molecule-1, MMP-9, IL-6, and TNF.14
These similarities between the antiinflammatory effects of statins and PPAR led us to investigate whether PPAR could mediate antiinflammatory effects of statins in vivo in models of acute inflammation and in vitro in macrophages and neutrophils.
Materials and Methods
Inflammation Tests
Subcutaneous dorsal pouches and carrageenan footpad edema were induced in C57BL6 wild-type and PPAR-null mice as described.16,17 Simvastatin at indicated doses or vehicle (CMC 0.5%) was given by gavage to mice 1 hour before inflammatory challenges (see the online data supplement available at http://circres.ahajournals.org).
Cell Culture
Lipopolysaccharide (LPS)-elicited neutrophils from air pouches and thioglycollate-elicited peritoneal macrophages were isolated as described.18 Cells were treated with the indicated reagents (see the online data supplement).
RNA Analysis
RNA extraction was performed using TRIzol reagent followed by reverse transcription (Invitrogen Life Technologies, Cergy-Pontoise, France). cDNA was quantified by real-time PCR on a MX4000 apparatus (Stratagene) using specific primers (see the online data supplement).
Kinase Assays and Immunoblot
After treatment, cells were washed with PBS and suspended in protein kinase C (PKC) lysis buffer, sonicated (Vibracell Hiddock 72442), and centrifuged at 4°C (3000 rpm, 15 minutes). Cell extracts (10 e) or cell extracteCimmunoprecipitated PKC (200 e) were incubated in kinase reaction buffer, histone H1 (1 e), or purified PPAR protein (400 ng) as substrates and (-32P)ATP (5 e藽i) (2000 Ci/mmol). Kinase reactions were performed as described previously.19 Immunoblots were performed using the Aurora detection system (ICN Pharmaceuticals, Orsay, France) (see the online data supplement).
Transient Transfections and Metabolic Labeling
COS-7 cells were transfected by lipofection with reporter and expression plasmids as indicated and incubated overnight with DMEM supplemented with 2% Ultroser. Cells were collected and luciferase and -galactosidase assays performed. For 35S-methionine labeling, cells were cultured in methionine-free minimum essential medium for 1 hour before supplementation with 35S-methionine (100 e藽i) for an additional 3 hours. For 33P-phosphate labeling, cells were deprived in phosphate-free minimum essential medium for 2 hours before supplementing the medium with 33P-phosphate (500 e藽i) for 5 hours, followed by PPAR immunoprecipitation (see the online data supplement).
Statistical Analysis
Statistical significance was determined using nonparametric ManneCWhitney or multivariate ANOVA tests followed by Scheffe post hoc or the unpaired t tests (transient transfections). Values of P<0.05 were considered as significant.
Results
PPAR Mediates the Acute Antiinflammatory Action of Simvastatin In Vivo
To investigate whether PPAR plays a role in inflammatory response modulation by statins in vivo, the influence of simvastatin was tested in wild-type and PPAR-null mice using 2 models of acute inflammation in which statins display antiinflammatory activity.16,17 Doses were chosen in accordance with these previous studies.16,17 The acute antiinflammatory action of simvastatin (10 to 50 mg/kg) administered orally 1 hour before LPS was first measured by the number of neutrophils recruited in air pouches by LPS.16 Simvastatin treatment decreased neutrophil recruitment in a dose-dependent manner (Figure 1A). Administration of a single dose of atorvastatin (30 mg/kg) exerted similar effects on neutrophil recruitment (not shown). Interestingly, the decrease of LPS-induced neutrophil recruitment by simvastatin was only observed in wild-type, but not in PPAR-null mice (Figure 1B). Similarly, in the carrageenan-induced footpad inflammation mouse model,17 a single dose of simvastatin given 1 hour before carrageenan injection blocked swelling only in wild-type, but not in PPAR-null mice (Figure 1C). These effects occurred independently of alterations in plasma lipid levels, because plasma cholesterol levels did not change after simvastatin treatment in either model (not shown). Thus, PPAR mediates the lipid-independent acute antiinflammatory activity of simvastatin in mice.
PPAR Mediates the Inhibition of LPS-Induced Inflammatory Response Genes by Simvastatin in Primary Macrophages and Neutrophils
Because neutrophils are a major cell type mediating the inflammatory response in these in vivo models, the expression of PPAR was analyzed in LPS-elicited neutrophils recovered from air pouches and compared with other cell types. PPAR mRNA levels were highest in neutrophils, whereas macrophages express similar levels as primary endothelial cells, a cell type in which the antiinflammatory effects of PPAR have been well documented20 (Figure 2A).
Subsequently, the role of PPAR in the modulation of inflammatory response gene expression (iNOS, TNF, or IL-6) by statins was investigated in macrophages and neutrophils isolated from wild-type and PPAR-null mice. Pretreatment (2 hours) of macrophages isolated from wild-type mice with a single dose of simvastatin, sufficient to inhibit HMG-CoA reductase activity,18 significantly decreased LPS-induced iNOS and IL-6 mRNA levels (Figure 2B and 2C). By contrast, simvastatin was without effect in macrophages isolated from PPAR-null mice. Similarly, pretreatment (2 hours) with simvastatin also significantly decreased LPS-induced iNOS and TNF mRNA levels in neutrophils isolated from wild-type but not from PPAR-null mice (Figure 2D and 2E). Simvastatin induced a dose-dependent decrease in iNOS mRNA and protein levels after LPS induction only in wild-type but not in PPAR-null macrophages (Figure 3A and 3B). Atorvastatin and fluvastatin pretreatment also decreased LPS-induced iNOS expression in a PPAR-dependent manner (Figure 3C and 3D). These data indicate that the antiinflammatory effect of statins on LPS-induced inflammatory response genes, such as iNOS, in macrophages and neutrophils is PPAR dependent.
Previous studies have shown that statins induce the expression of apolipoprotein A-I in human hepatoma HepG2 cells by modulating PPRE-dependent transcriptional activity of PPAR.21 To determine whether statins also regulate PPRE-dependent PPAR target genes in macrophages, the effects of simvastatin on the induction of CPT1 mRNA levels, a gene induced by PPAR in human primary macrophages,22 by the PPAR agonist GW9578 were investigated. As expected, GW9578 treatment (12 hours) increased CPT1 mRNA levels, but simvastatin treatment did not influence this induction (Figure 4A). By contrast, both simvastatin and GW9578 treatment decreased iNOS mRNA levels, and coincubation with both compounds resulted in a more pronounced inhibition of iNOS mRNA levels (Figure 4B). These results indicate that, unlike in hepatocytes, simvastatin selectively interferes in macrophages with PPAR inhibition of inflammatory response genes, likely by modulating PPAR-dependent transrepression activity.
The PKC Signaling Pathway Is Involved in LPS-Induced iNOS Expression and Is Inhibited by Simvastatin
Because the effect of simvastatin on LPS-induced iNOS expression occurs rapidly and requires PPAR, it was hypothesized that simvastatin exerts its effects via posttranslational modulation of PPAR activity. To determine which signaling pathway mediates LPS-induced iNOS expression in macrophages and neutrophils, the effects of different protein kinase inhibitors, which inhibit the PKC or MAPK signaling pathways, were tested. Incubation of macrophages with either the PKC inhibitor G6976, which selectively inhibits the Ca2+-dependent PKC and PKC isoforms, or the PKC inhibitor Ro318220, which inhibits all Ca2+-dependent PKC isoforms, prevented LPS-induced iNOS expression, whereas a MEK inhibitor U0126 was without effect (Figure 5A). Similarly, G6976 inhibited LPS-induced iNOS expression in neutrophils, whereas U0126 was without effect (Figure 5B).
To determine whether simvastatin modulates the Ca2+-dependent PKC signaling pathway in macrophages and neutrophils, its effect on the activity of PKC was investigated. PKC was immunoprecipitated from LPS-activated macrophages or neutrophils and in vitro phosphorylation experiments using purified histone H1 protein as substrate were performed. LPS treatment induced PKC activity in macrophages, whereas LPS-elicited neutrophils already displayed high basal PKC activity. Interestingly, pretreatment (2 hours) with simvastatin decreased PKC activity both in macrophages and neutrophils (Figure 5C and 5D). In addition, simvastatin treatment also decreased LPS-induced PKCII activity both in macrophages and neutrophils (not shown). Thus, inhibition of the Ca2+-dependent PKC signaling pathway by simvastatin could be involved in the effects of simvastatin on LPS-induced iNOS expression both in macrophages and neutrophils.
To determine whether the Ca2+-dependent PKC signaling pathway is involved in the PPAR-dependent inhibition of LPS-induced iNOS expression, the effect of G6976 on LPS-induced iNOS expression was investigated in macrophages isolated from wild-type and PPAR-null mice. Pretreatment (2 hours) of wild-type macrophages with G6976 resulted in a decrease of LPS-induced iNOS mRNA levels (Figure 6). By contrast, G6976 was without effect in macrophages isolated from PPAR-null mice. These results suggest a role for PPAR in the control of LPS-induced iNOS expression by the Ca2+-dependent PKC signaling pathway.
Simvastatin Decreases PPAR Phosphorylation by LPS-Activated PKC
To determine whether PKC modulates PPAR phosphorylation in macrophages and whether statins influence this phosphorylation, in vitro phosphorylation experiments using purified PPAR protein as substrate were performed using extracts from LPS-stimulated macrophages pretreated with simvastatin. Incubation with LPS induced the activity of kinases that phosphorylate PPAR in vitro. This effect was prevented by both simvastatin and G6976 (Figure 7A), whereas mPKCI, an inhibitor of all PKC isoforms, inhibited both basal and LPS-stimulated PPAR phosphorylation. Furthermore, PKC immunoprecipitated from LPS-activated macrophages was able to phosphorylate PPAR and this effect was inhibited by simvastatin pretreatment (Figure 7B). To confirm that simvastatin prevents PKC-induced PPAR phosphorylation in cells, metabolic labeling experiments were performed in PPAR and PKC-transfected COS cells. PKC induced the phosphorylation of PPAR, an effect that was inhibited by simvastatin (Figure 7C). Thus, LPS-induced PPAR phosphorylation occurs, at least partly, via PKC in macrophages and simvastatin inhibits this phosphorylation.
Simvastatin Increases PPAR Transrepression Activity on NFB via Its PKC Phosphorylation Sites
We have recently shown that the PKC signaling pathway modulates the transrepression activity of PPAR in hepatocytes via the PKC phosphorylation sites S179 and S230.19 Because the antiinflammatory action of PPAR is, at least partly, mediated by the repression of NFB transcriptional activity via direct interaction with NFB-p65 protein,23,24 it was investigated whether phosphorylation of PPAR on its PKC sites modulates its transrepression activity on NFB-p65. To eliminate confounding effects of NFB-activating pathways, PPAR activity was directly tested on nuclear-activated NFB by using a chimeric protein composed of the yeast GAL4 DNA binding domain fused to p65 and a reporter vector driven by a GAL4 response element.23 Cells were cotransfected with increasing concentrations of PPAR wild type or PPAR mutated in its PKC phosphorylation sites, PPAR(S179A-S230A), a nonphosphorylatable, nonphosphomimetic mutant. At all concentrations tested, PPAR(S179A-S230A) induced a more pronounced inhibition of p65-driven reporter activity compared with wild-type PPAR (Figure 8A), suggesting that PKC phosphorylation of PPAR inhibits PPAR transrepression activity on NFB.
To demonstrate a role for PKC in the transrepression activity of PPAR on NFB-p65, a constitutive active form of PKC (CA-PKC) was tested on p65 transrepression by wild-type PPAR or PPAR(S179A-S230A). To obtain optimal sensitivity, wild-type PPAR was transfected at a concentration ratio exerting clear, basal repression of p65-driven transcriptional activity. Under these conditions, cotransfection of CA-PKC prevented repression of p65-driven transcriptional activity by wild-type PPAR. By contrast, CA-PKC did not repress the activity of PPAR(S179A-S230A) (Figure 8B).
Finally, to determine whether statin treatment modulates PPAR transrepression activity on NFB via its PKC phosphorylation sites, the effect of simvastatin was tested on p65 transrepression by wild-type PPAR or PPAR(S179A-S230A). In this experiment, PPAR wild type was transfected at a concentration ratio that does not yet influence p65-driven transcriptional activity. Under these conditions, incubation with simvastatin induced a significantly more pronounced inhibition of p65-driven transcriptional activity in the presence of wild-type PPAR, an effect that was not observed with PPAR(S179A-S230A) (Figure 8C). These results indicate that inhibition of PKC by simvastatin enhances PPAR transrepression activity on NFB.
Discussion
Clinical trials and in vitro studies have shown that statins and PPAR agonists share antiinflammatory properties by regulating inflammatory-response genes.14,25 In the present study, using 2 well-characterized animal models of acute inflammation, we demonstrate that simvastatin requires PPAR expression to exert its antiinflammatory effects in vivo. The in vivo antiinflammatory effects of simvastatin on footpad swelling and neutrophil recruitment in air poucheCbearing mice already occur within 1 hour after a single oral administration, indicating that the PPAR-dependent antiinflammatory effects of simvastatin occur rapidly. The observed effects, therefore, cannot be explained by the plasma lipid-lowering activities of the drug. Indeed, as previously shown,17 simvastatin treatment did not change lipid levels in mice (not shown). Thus, simvastatin exerts direct antiinflammatory effects via PPAR, independent of its plasma cholesterol-lowering activities.
The involvement of PPAR in the antiinflammatory effects of statins is further evidenced in vitro in experiments with primary macrophages and neutrophils, 2 cell types mediating acute inflammatory responses. PPAR activators act on a variety of vascular cells such as endothelial cells (ECs), vascular smooth muscle cells (VSMCs), monocytes/macrophages, and T cells, which all express PPAR.14 Although PPAR mRNA is expressed at low levels in peritoneal macrophages, it clearly plays a functional role in the antiinflammatory effects of simvastatin in vitro and in vivo, as evidenced by the lack of simvastatin effects on PPAR-deficiency. These results are consistent with a recent study showing that PPAR prevents macrophage foam cell formation in the peritoneal cavity.26 In addition, we show that neutrophils express high levels of PPAR mRNA. In these cells, PPAR also mediates the antiinflammatory effects of simvastatin, thus identifying a novel cell type in which PPAR exerts antiinflammatory activities.
To our knowledge, this is the first demonstration of the existence of a cross-talk between statins and PPAR in the regulation of lipid-independent inflammatory responses. These results thus extend previous studies on liver and lipid metabolism,21,27,28 indicating that both hypolipidemic and antiinflammatory effects of statins could involve PPAR.
We provide molecular evidence that statins modulate Ca2+-dependent PKC signaling pathway in macrophages and neutrophils resulting in PPAR-dependent inhibition of LPS-induced inflammatory response genes, such as iNOS. These results are of particular interest because PKC plays a role in the inflammatory response. In vivo overexpression of PKC in the epidermis results in severe neutrophil-mediated inflammation.29 In vitro in macrophages, PKC regulates LPS-induced iNOS, TNF, and IL-1 expression.30 Our results suggest the implication of other Ca2+-dependent PKC isoforms, such as PKCII, in the PPAR-dependent antiinflammatory effect of statins, because simvastatin inhibits also LPS-induced PKCII activity in macrophages and neutrophils (not shown) and because PKCII also phosphorylates PPAR on its PKC phosphorylation sites S179-S230 in vitro.19 The mechanism underlying the activation of PKC by LPS and the inhibitory effect of simvastatin on LPS-induced PKC activation in macrophages and neutrophils is presently unclear. Our results suggest that LPS induces PKC translocation to the cell membrane in macrophages, but not in neutrophils, and that simvastatin may block this effect (not shown). It will be of interest to determine whether PKC activators (PLC, PDK1) or repressors (DAGK or PP1 phosphatases)31 are regulated by simvastatin in these cells.
We previously demonstrated that classical PKCs phosphorylate PPAR in vitro.19 Here, we show that LPS induction of PKC in macrophages results in increased PPAR phosphorylation in vitro and that PKC overexpression increased PPAR phosphorylation in cells. Moreover, simvastatin inhibited PKC-induced PPAR phosphorylation. PPAR is a phosphoprotein phosphorylated by different kinases, such as extracellular signal-regulated kinase,32 p38,33 and PKA.34 Previously identified PPAR-phosphorylating kinases all enhanced PPAR transcriptional activity. In our report, we show, by using PPAR mutated on its PKC phosphorylation sites (S179-S230) as well as a CA-PKC, that activated PKC inhibits the transrepression properties of PPAR on NFB-p65. By contrast, simvastatin enhances PPAR transrepression activity acting via its PKC phosphorylation sites (S179-S230), suggesting that simvastatin stimulates PPAR transrepression activity via inhibition of PPAR inactivation by PKC. Whereas in liver cells, the PKC signaling pathway also regulates the ligand-dependent PPAR transactivation properties, as demonstrated by enhanced CPT1 induction,19 in macrophages, simvastatin treatment did not modify PPAR-induced CPT1 expression, even in the presence of a PPAR agonist. Inhibition of the Ca2+-dependent PKC signaling pathway by simvastatin thus only influences the transrepression properties of PPAR in macrophages. We propose that activation of PKC by inflammatory stimuli, such as LPS, leads to the phosphorylation and subsequent deactivation of PPAR. Statins prevent PKC activation by LPS and, as a consequence, inhibit PPAR phosphorylation by PKC, leading to enhanced PPAR transrepressive activity on NFB (Figure 8D).
The effects of statins on inflammation could also involve NFB-independent mechanisms, eg, via modulation of CD62L and CD11b adhesion molecule expression in monocytes.35 However, we did not observe any effect of statins on the expression of these adhesion molecules in neutrophils (not shown). Nonetheless, our results do not exclude that other PPAR- and PKC-independent mechanisms contribute also to the antiinflammatory effects of statins because statins regulate other signaling pathways such as phosphatidylinositol 3-kinase and mitogen-activated protein kinase.36,37
Macrophages and neutrophils are mediators of the early inflammatory response that play a major role in the inflammation and tissue damage associated with both infectious and noninfectious diseases, such as sepsis, acute coronary syndrome, rheumatoid arthritis, and ischemic stroke.38eC41 Results from basic research and clinical trials indicate that the pleiotropic antiinflammatory effects of statins may result in clinical benefit in such inflammatory diseases.42eC44 Our results demonstrating that statins exert their antiinflammatory effects through PPAR provide further evidence for the importance of such pleiotropic activities. Clinical studies with PPAR agonists have shown significant protective effects against CVD and stroke, effects that cannot be attributed to their cholesterol-lowering activities alone.14,15 Our results thus provide a potential clinically relevant mechanism for the pleiotropic effects of statins through PPAR.
Acknowledgments
This work was supported by grants from the Fondation pour la Recherche Medicale (to R.P. and C.B.), Institut de France (O.B.), Fondation Leducq, and European community grant QLRT-1999-01007. We thank Drs Parker and Haegeman for providing expression plasmids.
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