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INFLAMMATION AND IMMUNOPHARMACOLOGY
Is Not Necessary for Synthetic PPAR Agonist Inhibition of Inducible Nitric-Oxide Synthase and Nitric Oxide
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina (M.B.C., J.L.S., J.Z., G.S.G.); Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (C.J.N., F.J.G.); and Medical Research Service, Ralph H. Johnson VA Medical Center, Charleston, South Carolina (G.S.G.)
Received July 15, 2004; accepted September 8, 2004.
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Abstract |
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agonists inhibit inducible nitric-oxide synthase (iNOS), tumor necrosis factor-
, and interleukin-6. Because of these effects, synthetic PPAR agonists, including thiazolidinediones, are being studied for their impact on inflammatory disease. The anti-inflammatory concentrations of synthetic PPAR agonists range from 10 to 50 µM, whereas their binding affinity for PPAR is in the nanomolar range. The specificity of synthetic PPAR agonists for PPAR at the concentrations necessary for anti-inflammatory effects is thus in question. We report that PPAR is not necessary for the inhibition of iNOS by synthetic PPAR agonists. RAW 264.7 macrophages possess little PPAR, yet lipopolysaccharide (LPS)/interferon (IFN)-induced iNOS was inhibited by synthetic PPAR agonists at 20 µM. Endogenous PPAR was inhibited by the transfection of a dominant-negative PPAR construct into murine mesangial cells. In the transfected cells, synthetic PPAR agonists inhibited iNOS production at 10 µM, similar to nontransfected cells. Using cells from PPAR Cre/lox conditional knockout mice, baseline and LPS/IFN-induced nitric oxide levels were higher in macrophages lacking PPAR versus controls. However, synthetic PPAR agonists inhibited iNOS at 10 µM in the PPAR-deficient cells, similar to macrophages from wild-type mice. These results indicate that PPAR is not necessary for inhibition of iNOS expression by synthetic PPAR agonists at concentrations over 10 µM. Intrinsic PPAR function, in the absence of synthetic agonists, however, may play a role in inflammatory modulation.
, known as thiazolidinediones (TZDs), are currently being used for treatment of type II diabetes. Their clinical efficacy in diabetes coupled with the early reports of their effect on inflammatory mediator reduction in macrophage cell lines has lead to the study of PPAR as an endogenous modulator of inflammation. The intrinsic ligand for PPAR remains unclear. A prostaglandin J2 metabolite was proposed to be an endogenous PPAR agonist. This metabolite was shown effective at reducing markers of inflammation, such as inducible nitric-oxide synthase (iNOS), at concentrations less than 10 µM (Jiang et al., 1998
; Ricote et al., 1998; Petrova et al., 1999). However, it was later shown that in addition to binding to PPAR, 15-deoxy-
12,14-prostaglandin J2 (15dPGJ2) also stabilized NF-
B and prevented its activation of the iNOS promoter (Straus et al., 2000). A reactive metabolite, 15dPGJ2 is capable of donating an electron to the NF-B. Recently, the lack of physiologic relevance of 15dPGJ2 as an endogenous PPAR agonist was demonstrated, and toxicity of the compound limits its clinical utility (Bell-Parikh et al., 2003). Thus, it is clear from a number of studies that PPAR is not necessary for the anti-inflammatory properties of 15dPGJ2. This same PPAR-independent reactivity has not been reported for the synthetic PPAR agonists such as the TZDs that are known to be clinically well tolerated. In most inflammatory studies, PPAR agonist concentrations above 20 µM were necessary to see any effect. This is a concern because the Kd or binding affinity for most synthetic PPAR agonists is in the nanomolar range (Oates et al., 2002).
The reason for the discrepancy between the Kd and effective doses for these synthetic PPAR agonists is unknown. Concentrations of synthetic PPAR agonists above 20 µM approach the upper limits of physiological relevance and yet are necessary to reduce inflammatory mediator production in macrophage or lymphocyte cell lines or to induce apoptosis in cancer cell lines (Elstner et al., 1998; Clark et al., 2000; Schlezinger et al., 2002). These studies raise the question as to whether PPAR is the only anti-inflammatory target of these synthetic compounds. Although the TZDs may be binding to PPAR, they may also be binding to other receptors at the necessary high doses reported in many publications, including ours (Reilly et al., 2000, 2001; Oates et al., 2002).
Synthetic PPAR agonists are inhibitors of iNOS, nitric oxide (NO), tumor necrosis factor-, and interleukin-6 (Jiang et al., 1998; Ricote et al., 1998; Alleva et al., 2002). Because of these early reports, the TZDs and other synthetic PPAR agonists are being studied in both animal models and human patients for their effects on inflammatory diseases such as rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis (Kawahito et al., 2000; Reilly et al., 2000, 2001; Lewis et al., 2001; Oates et al., 2002; Duvanel et al., 2003; Kielian and Drew, 2003). The reported effective concentrations of various PPAR agonists in these reports range from 10 to 50 µM.
In the experiments reported herein, we determined the necessity of PPAR for blocking iNOS activation by synthetic PPAR agonists. RAW 264.7 macrophages possess little to no PPAR, yet synthetic PPAR agonists inhibited lipopolysaccharide (LPS)/interferon (IFN)-induced iNOS and NO. We then inhibited the function of endogenous PPAR in murine mesangial cells with the addition of a dominant-negative (D/N) PPAR construct. Despite this inhibition, synthetic PPAR agonists were still capable of inhibiting induced iNOS and NO production. Finally, we used a PPAR-conditional knockout mouse to demonstrate that the removal of PPAR in macrophages derived from these mice had no effect on the ability of these agonists to inhibit induced iNOS and NO production. Together these results indicate that PPAR is not necessary for the reduction of NO by synthetic PPAR agonists. The discovery of the additional anti-inflammatory targets of these drugs will be important for the design of more specific and active compounds that could be used to suppress inflammation.
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Materials and Methods |
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was purchased from Pierce Endogen (Rockford, IL). GW347845X (GW) was a gift from Kathleen Brown (GlaxoSmith-Kline, Research Triangle Park, NC). The GW7845 compound is a non-TZD PPAR agonist (Cobb et al., 1998), and it has been shown to be more potent at producing adipocyte differentiation than the TZDs rosiglitazone and pioglitazone (Suh et al., 1999a,b). Pioglitazone and rosiglitazone are both TZDs that were gifts from Yan Huang (Department of Medicine, Division of Endocrinology, Medical University of South Carolina, Charleston, SC).
Mice and Macrophage Harvest. PPAR conditional null mice were generated using the Cre-loxP system where exon 2 of the PPAR gene is removed once induction of the Cre recombinase takes place according to the design of Akiyama et al. (2002). To induce PPAR deletion, we followed the procedure of Akiyama et al. (2002). Briefly, we induced the knockout through a series of five polyinosinic-polycytidylic acid (pIpC) intraperitoneal injections into the PPAR conditional knockout mice. The injections were spaced 4 days apart.
Peritoneal macrophages were elicited with thioglycollate (Sigma-Aldrich). A 3% thioglycollate solution was injected intraperitoneally, and 4 days later, the macrophages were harvested through phosphate-buffered saline lavage. Ten milliliters of ice-cold phosphate-buffered saline was injected into the peritoneal cavity and gently removed. Once collected, the cells were spun down and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. The cells were counted and plated at 1 million cells/well into six-well plates for further stimulation as described in the results.
Transcription Assays. Mesangial cells were transiently transfected with either a PPAR D/N and/or a PPAR response element (PPRE) vector, PPRE-luciferase, and a Renilla luciferase construct using FuGENE 6 (Roche Diagnsotics, Nutley, NJ) per manufacturer's instructions. The PPAR dominant-negative construct has been described previously (Gurnell et al., 2000). Briefly, the charge-clamp site in the AF-2 region was mutated to prevent coactivator recruitment while maintaining ligand and DNA binding (Gurnell et al., 2000). The D/N construct was a gift from Floyd Chilton and Carl Clay (Wake Forest University, Winston-Salem, NC). The PPRE-luciferase construct was a gift from Bruce Spiegelman (Dana-Farber Cancer Institute, Boston, MA), and the Renilla luciferase plasmid was purchased from Promega (Madison, WI).
Mesangial cells transfected with the luciferase constructs were analyzed using the dual luciferase assay (Promega) on a Luminoskan Ascent (ThermoLabSystems, Baltimore, MD) following manufacturer's instructions. PPRE-luciferase expression was normalized to the constitutive Renilla luciferase expression values.
Nitrite/Nitrate Analysis. Stimulated cell supernatants were filtered in 10,000 mol. wt. Ultrafree-MC centrifuge tubes (Millipore Corporation, Billerica, MA). Nitrate was converted to nitrite using nitrate reductase (Roche Diagnostics, Indianapolis, IN) as described previously (Granger et al., 1996). The Griess reaction was then performed on all samples and standards to allow for absorbance analysis at 540 nM by a TiterTek Multiskan/MCC 340 plate-reader (ThermoLabsystems).
RT-PCR. Total RNA was isolated with RNeasy (QIAGEN, Valencia, CA) and quantitated on an Eppendorf biophotometer. One hundred nanograms of RNA were reverse-transcribed using the Moloney murine leukemia virus reverse transcriptase (Ambion, Austin, TX). PPAR and iNOS RNA levels were assessed through PCR. The primers for PPAR were as follows: forward, 5'-TGAGGAGAAGTCACACTCTG-3'; and reverse, 5'-TGGGTCAGCTCTTGTGAATG-3' (Clark et al., 2000). The cycling conditions were as follows: 35 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 45 s, followed by a final extension step at 72° for 10 min. Murine iNOS transcript levels were assessed using the Gene-Specific Relative RT-PCR kit for murine iNOS (Ambion). Cycling conditions were optimized according to manufacturer's instructions. Both PPAR and iNOS transcript levels were normalized to 18S as an internal control. Polymerase chain reaction was performed on an iCycler (Bio-Rad, Hercules, CA). Bands were UV visualized in a 2% agarose gel containing 0.005% ethidium bromide.
Protein Analysis. Stimulated cell lysates were harvested and analyzed for protein content utilizing the Bradford assay (Bio-Rad). Forty micrograms of protein was loaded onto a 4 to 15% Tris-HCl Criterion gel (Bio-Rad) and electrophoresed at 250 V in SDS/Tris-glycine running buffer. The samples were then transblotted to polyvinylidene difluoride membranes on a Criterion Transblot apparatus for 30 min at 400 mA (Bio-Rad). The membranes were then blocked in 5% milk/Tris-buffered saline/1% Tween 20. The primary antibodies were as follows: PPAR polyclonal antibody at 1:400 (Cayman Chemical, Ann Arbor, MI), PPAR
polyclonal antibody at 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), iNOS polyclonal antibody at 1:2500 (BD Transduction Laboratories, Lexington, KY), and FLAG-AP-conjugated monoclonal antibody at 1:300 (Sigma-Aldrich). For PPAR and iNOS, the secondary antibody used was goat anti-rabbit IgG-AP labeled (Southern Biotechnology Associates, Birmingham, AL). For PPAR, the secondary was a rabbit anti-goat IgG-AP (Sigma-Aldrich). The membranes were exposed to enhanced chemifluorescence substrate and analyzed on a STORM 860 phospho/fluorimager. Equal protein-loading was reconfirmed by Coomassie Blue staining of the membrane (data not shown).
Statistical Analysis. Two-way analysis of variance was used to determine statistical significance between groups. GraphPad Prism software was used for analysis (GraphPad Software Inc., San Diego, CA).
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Results |
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Agonists in PPAR-Deficient RAW 264.7 Macrophages. RAW 264.7 macrophages contain very low to undetectable amounts of PPAR (Ricote et al., 1998; Castrillo et al., 2001). We stimulated RAW 264.7 macrophages with LPS and IFN to induce NO and iNOS production and added increasing doses of GW347845X, rosiglitazone, or pioglitazone to the cultured cells. Induced NO production was inhibited by all three PPAR agonists. NO was inhibited at 20 µM pioglitazone and GW347845X and at 30 µM rosiglitazone in stimulated RAW 264.7 macrophages (Fig. 1A). There was no effect of these concentrations on cell viability as determined by trypan blue exclusion. iNOS was inhibited at both the RNA and protein level in the stimulated cell line at 20 µMby all three compounds (pioglitazone treatment is not shown, but was similar) (Fig. 1, B and C). We performed Western blots and RT-PCR to confirm the absence of PPAR in the RAW 264.7-macrophage cell line. Figure 2 demonstrates the lack of PPAR at both the RNA and protein levels in the RAW 264.7 macrophage cell line with and without stimulation.
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A Dominant-Negative PPAR Construct Has No Effect on the Ability of PPAR Agonists to Inhibit iNOS and NO. We transiently transfected murine mesangial cells with a FLAG-tagged D/N PPAR construct and stimulated them with LPS/IFN and increasing doses of PPAR agonists. The D/N construct was mutated in the AF-2 region to prevent coactivator recruitment and corepressor release. The D/N still binds the ligand and retains an intact DNA-binding region and an active ligand-independent AF-1 transactivation domain. Despite the D/N inhibiting PPAR activity, mesangial cells were just as responsive to the PPAR agonists as nontransfected cells. Both NO production and iNOS expression were inhibited at 5 to 10 µM GW347845X and 10 to 20 µM rosiglitazone or pioglitazone (Figs. 3 and 4).
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We confirmed the presence of PPAR and the transfected FLAG-tagged D/N construct by anti-PPAR and anti-FLAG Western blots. The FLAG antibody detected the heavier FLAG-tagged PPAR in the D/N-transfected cells, which verified a successful transfection (Fig. 5A). The PPAR antibody bound both endogenous PPAR and the transfected FLAG-tagged D/N PPAR (Fig. 5B).
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We verified the reduced activity of PPAR in the D/N-transfected cells via a luciferase assay. PPAR D/N-transfected cells were cotransfected with a PPRE-luciferase construct and Renilla luciferase construct as described under Materials and Methods. Once stimulated with the PPAR agonist GW347845X, only cells that did not contain the PPAR D/N construct were able to produce a significant stimulatory response (Fig. 5B). This experiment was also performed with both rosiglitazone and pioglitazone with similar results (data not shown). Although there was a slight increase in luciferase expression with the addition of the PPAR agonist in the PPAR D/N-transfected cells, there was no dose-response and the values were significantly lower from cells without the D/N transfection. These results indicate that PPAR activity was adequately reduced by the transfected PPAR D/N construct.
PPAR–/–Macrophage iNOS and NO Expression Are Inhibited by PPAR Agonists. To further confirm that PPAR agonists suppress iNOS in the absence of PPAR, macrophages elicited from PPAR conditional knockout mice were used. Mice heterozygous or homozygous for the presence of the Cre enzyme will have their PPAR gene disrupted when treated with pIpC. Mice that are Cre–/–maintain an intact PPAR gene even when treated with pIpC.
Peritoneal macrophages harvested from Cre+ and Cre–/– mice treated with pIpC were cultured and stimulated with inflammatory mediators and increasing doses of PPAR agonists GW347845X, rosiglitazone, or pioglitazone. NO and iNOS were induced with LPS/IFN stimulation. Both NO and iNOS were inhibited at 10 µM GW347845X in both the Cre+/pIpC and Cre–/pIpC derived macrophages (Figs. 6A and 7A). Rosiglitazone and pioglitazone required 20 µM to inhibit NO and iNOS in the LPS/IFN-stimulated macrophages (Figs. 6, B and C, and 7, B and C). NO production was significantly increased both at baseline and with LPS/IFN stimulation in cells lacking PPAR compared with cells expressing PPAR. Despite the baseline and induced increase in NO production in the PPAR-deficient cells, iNOS and NO production in both cell lines were inhibited by PPAR agonists. We confirmed the lack of a functional PPAR protein through Western blot. The Cre–/pIpC macrophages expressed PPAR, whereas the Cre+/pIpC macrophages had barely perceptible levels of PPAR protein (Fig. 8). These results indicate that synthetic PPAR agonists inhibit iNOS expression and NO production in the absence of PPAR, although endogenous PPAR seems to modulate baseline and induced NO production.
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PPAR Protein Expression Is Stable with the Addition of TZDs. Since PPAR activation by TZDs has also been shown, we assessed whether PPAR expression was altered with the TZDs or Cre induction in the knockout macrophages. Peritoneal macrophages were harvested as described above and treated with the inflammatory mediators LPS/IFN and increasing concentrations of pioglitazone, rosiglitazone, and the GW compound as described above. PPAR protein expression was constant despite increasing levels of the TZDs and induction of the PPAR knockout (Fig. 9). Only the data for pioglitazone are shown, but results were similar for rosiglitazone and GW347845X. Additionally, PPAR expression was detected in both the RAW macrophages and mesangial cells (data not shown).
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Discussion |
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agonists are dependent on the presence of PPAR.In the RAW 264.7 macrophage cell line and PPAR conditional knockout-derived macrophages, GW347845X, rosiglitazone, and pioglitazone inhibited iNOS expression at the RNA and protein level while also reducing NO production despite the absence of PPAR. In mesangial cells transiently transfected with a dominant-negative PPAR construct, the PPAR agonists again effectively inhibited iNOS expression and NO production, despite reduced PPAR activity. Mesangial cells were used due to their ease in transfection compared with macrophages. These experiments also indicate that the PPAR-independent effects of the synthetic PPAR agonists are not macrophage-specific. Thus, our results demonstrate that PPAR is not necessary for synthetic PPAR agonists to inhibit NO production in murine macrophages and mesangial cells.
Although 15dPGJ2 is well known via published studies to act independently of PPAR, the synthetic PPAR agonists were previously thought to be exerting their anti-inflammatory effects solely through PPAR activation. In some case, these compounds were designed to specifically interact with the PPAR ligand-binding site. The results reported herein will require that future studies using high concentrations of these compounds will need to prove that the biological effect noted is mediated solely by PPAR. PPAR's apparent endogenous role in controlling inflammation without the addition of exogenous synthetic compounds is also a novel finding of potential clinical relevance. Discovery of the yet unknown true endogenous ligand of PPAR will aid in elucidating the intrinsic role of PPAR in controlling inflammation.
The presence of PPAR is clearly necessary for certain cellular functions and development as evidenced by the embryonic lethality of PPAR–/– mice (Barak et al., 1999). Akiyama et al. (2002) showed that the loss of PPAR inhibited gene expression of lipoprotein lipase, CD36, LXR, ABCG1, and apolipoprotein E, which are vital for the control of cholesterol efflux from macrophages. However, Akiyama et al. (2002) also showed that troglitazone was able to inhibit ABCA1 gene expression in PPAR-deficient macrophages, whereas other TZDs required PPAR for inhibition of this gene. Troglitazone is known to have nonspecific effects and is no longer used clinically due to toxic side effects (de Dios et al., 2001).
Our investigations were initiated due to the discrepancy between the binding affinities of clinically available synthetic PPAR agonists for PPAR and the concentrations necessary for anti-inflammatory effects. The reported binding affinities for various PPAR agonists to PPAR are log range lower than the concentrations necessary for iNOS and NO inhibition or apoptotic inducement (Oates et al., 2002). It is possible that these high concentrations are necessary to achieve intracellular levels required for accessing PPAR in the nucleus. Recently, McIntyre et al. (2003) demonstrated that lysophosphatidic acid (LPA) is a potent PPAR agonist. Importantly, LPA is able to traverse both the cellular and nuclear membranes and activates PPAR at a concentration around 2 µM, which is similar to its serum concentration (McIntyre et al., 2003). Although McIntyre et al. (2003) showed that LPA activates PPAR, which subsequently initiates CD36 activation, it has not been determined whether LPA has any effect on inflammatory mediators, such as iNOS and NO.
In previous studies by our group, mesangial cells derived from MRL/lpr mice (lupus mouse model) had less PPAR expression than BALB/c derived mesangial cells (Reilly et al., 2001). Baseline and stimulated NO production was higher in MRL/lpr mesangial cells than BALB/c cells, perhaps reflecting the decreased expression of PPAR. However, the MRL/lpr mesangial cells were just as responsive to TZD treatment for iNOS inhibition as the BALB/c mesangial cells (Reilly et al., 2000). The equivalent response to PPAR agonists, despite striking differences in PPAR expression, supports our hypothesis that PPAR is not necessary for the inhibition of iNOS by synthetic agonists.
Macrophage differentiation was also hypothesized to require the presence of PPAR (Ricote et al., 1999); however, PPAR–/–embryonic stem cells were able to be induced to become macrophages in vitro (Chawla et al., 2001; Moore et al., 2001). Although PPAR may not be necessary for macrophage development and synthetic ligand-induced iNOS reduction, it may still play a key role in endogenous inflammatory control as evidenced by the heightened baseline and post stimulatory NO production in PPAR–/–macrophages.
A mechanism for PPAR-dependent inhibition of iNOS has been proposed previously. Li et al. (2000) described the transrepression mechanism where PPAR competes with NF-B for coactivators necessary for iNOS transcription. To achieve repression, PPAR must be highly activated to remove enough coactivators from the available pool to inhibit NF-B-mediated activation of the iNOS gene. The D/N construct used herein was designed to ineffectively recruit coactivators, yet the PPAR agonists were still effective inhibitors of NO and iNOS. Thus, these data indicate that transrepression acting through PPAR was not playing an important role in the suppression of iNOS transcription by the PPAR agonists. Transrepression through another nuclear hormone receptor stimulated by the agonists, however, may still be operational.
In studies related to ours, Castrillo et al. (2001) reported that the non-TZD PPAR agonist L-796,449 inhibited NO production independently of PPAR in RAW 264.7 macrophages. They reported, however, that L-796,449 functions similarly to 15dPGJ2 via stabilization of IB by inhibition of IB kinase complex. This IB stabilization likely explains the non-PPAR-mediated inhibition of NO by this compound. We have shown previously, as have others, that TZDs do not impact localization of NF-B to the nucleus or stabilize IB (Reilly et al., 2001; Guyton et al., 2003).
Because of the similarities between nuclear hormone receptors, it is possible that a related family member may also be binding the TZDs and non-TZD PPAR agonists. PPAR seems a likely possibility; however, published evidence indicates that PPAR agonists at high concentrations do not yield the same profile of anti-inflammatory effects as PPAR agonists (Delerive et al., 1999). Welch et al. (2003) demonstrated via microarray that at high concentrations rosiglitazone inhibited induction of LPS target genes in PPAR-deficient macrophages, possibly through PPAR. These results were not confirmed at the protein level and required 20 µM or higher concentrations to achieve significant inhibition. PPAR expression remains constant in the cell lines reported here and therefore in a PPAR-deficient cell line, it may respond to PPAR agonists (Berger and Moller, 2002). Some PPAR agonists have cross-reactivity with PPAR, with different effects depending on the receptor activated (Berger et al., 1999). In foam cells associated with atherosclerotic lesions, agonists of PPAR promoted lipid uptake and efflux. Deletion of PPAR from the foam cell macrophages increased the availability of inflammatory suppressors, which in turn reduced atherosclerotic lesions by more than 50% (Lee et al., 2003).
Despite the apparent PPAR-independent effects of the PPAR agonists, the increased baseline and induced NO production by PPAR-deficient macrophages compared with controls suggests endogenous PPAR expression may play a modulating role in inflammatory mediator production. The presence of functional PPAR seems to modulate baseline NO production as well as the amount of NO produced after an inflammatory stimulus. The in vivo relevance of this endogenous PPAR inflammation control is unclear at present. Synthetic PPAR agonists were effective in reducing inflammation in ulcerative colitis and atherosclerosis (Su et al., 1999; Pasceri et al., 2000; Lewis et al., 2001). Mice that are PPAR knockout heterozygotes are more susceptible to collagen-induced arthritis and experimental autoimmune encephalitis than wild-type littermates, indicating a clinically relevant intrinsic role for PPAR in the control of inflammation (Bright et al., 2003; Cuzzocrea et al., 2003). We believe the decreased expression of PPAR by mesangial cells from MRL/lpr mice may explain the increased baseline and induced NO production by these cells compared with controls.
In summary, we have demonstrated that PPAR is not necessary for synthetic TZD and non-TZD PPAR agonist reduction of iNOS expression and NO production in macrophages and mesangial cells. Although not necessary, PPAR may be sufficient in certain cell lines to mediate a reduction of iNOS expression and NO production at baseline and after inflammatory stimulation. It will be important to discover the alternative target of these PPAR agonists. Once this target is known, it may be used to design more specific drugs for reducing inflammatory mediators in diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.
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Footnotes |
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ABBREVIATIONS: PPAR, proliferation-activated receptor; TZD, thiazolidinedione; 15dPGJ2, 15-deoxy-12,14-prostaglandin J2; NF-B, nuclear factor-B; iNOS, inducible nitric-oxide synthase; NO, nitric oxide; LPS, lipopolysaccharide; IFN, interferon; D/N, dominant-negative; pIpC, polyinosinic-polycytidylic acid; PPRE, proliferation-activated receptor response element; RT-PCR, reverse transcription-polymerase chain reaction; LPA, lysophosphatidic acid; IB, inhibitor B; L-796,449, 3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio) phenylacetic acid.
Address correspondence to: Dr. Gary S. Gilkeson, Division of Rheumatology and Immunology, Medical University of South Carolina, 96 Jonathon Lucas St., Suite 912, P.O. Box 25063, Charleston, SC 29425. E-mail: gilkeson{at}musc.edu
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