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J. Biol. Chem., Vol. 276, Issue 36, 34082-34088, September 7, 2001

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Peroxisome Proliferator-activated Receptor--independent Inhibition of Macrophage Activation by the Non-thiazolidinedione Agonist L-796,449

COMPARISON WITH THE EFFECTS OF 15-DEOXY-^12,14-PROSTAGLANDIN J₂^*

Antonio Castrillo^§, Marina Mojena^§¶, Sonsoles Hortelano, and Lisardo Boscá

From the  Instituto de Bioquímica (Centro Mixto CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain, and ^¶ Centro de Investigación Básica de España (CIBE), Merck, Sharp and Dohme, Josefa Valcarcel 38, 28027 Madrid, Spain

Received for publication, March 20, 2001, and in revised form, June 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The effects of L-796,449 (3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic acid; referred to henceforth as compound G), a thiazolidinedione-unrelated peroxisome proliferator activated-receptor- (PPAR-) agonist, on early signaling in lipopolysaccharide-activated RAW 264.7 macrophages were analyzed and compared with those elicited by 15-deoxy-^12,14-prostaglandin J₂ and the thiazolidinedione rosiglitazone. Compound G inhibited the activation of nuclear factor B through the impairment of the targeting and degradation of IB proteins and promoted a redistribution of IB and IB in the nucleus of activated cells. Compound G inhibited IB kinase (IKK) activity both in vivo and in vitro, suggesting a direct mechanism of interaction between this molecule and the IKK complex. The effect of compound G on IKK activity was independent of PPAR- engagement because RAW 264.7 cells expressed negligible levels of this nuclear receptor, and rosiglitazone failed to mimic these actions. Moreover, treatment of activated macrophages with compound G enhanced the synthesis of superoxide anion, which, in combination with the NO produced under activation conditions, triggered apoptosis through the intracellular synthesis of peroxynitrite. These results suggest that compound G might contribute to the resolution of inflammation by favoring the induction of apoptosis through mechanisms independent of PPAR- engagement.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptors (PPARs)¹ are liganddependent nuclear transcription factors that have been implicated in the regulation of lipid and glucose metabolism, morphogenesis, and cell growth, differentiation, and homeostasis (1-4). This family of nuclear receptors consists of three members encoded by distinct genes, leading to the expression of PPAR-, PPAR-, PPAR-1, and PPAR-2, with the latter two isoforms resulting from the presence of alternative promoters in the 5'-flanking region of the gene (3, 5). Moreover, deficient signaling through these receptors is involved in the onset of various metabolic diseases such as diabetes, obesity, and atherosclerosis (6-9). PPARs exhibit specific patterns of cellular distribution: whereas PPAR- is expressed in tissues with high catabolic rates of fatty acids, such as the liver, muscle, and heart (4, 10-12), PPAR- expression is more restricted to adipose tissue and to selected cells of the immune system such as macrophages, where it is induced during the extravasation process of monocytes from the blood flow, especially in the course of activation by pro-inflammatory stimuli (6, 7, 13, 14). Both PPAR- and PPAR- have been identified as important regulators of the immune response. In macrophages, activated PPAR- acts as a transrepressor inhibiting the expression of genes requiring the binding of NF-B, activator protein-1, and signal transducer and activator of transcription-1 to their promoter regions (15, 16) . In addition to this, a role for PPAR- in inflammation was deduced from animals lacking this receptor because they exhibited prolonged responses to pro-inflammatory cytokines (1). Moreover, activation of PPAR- and PPAR- has been shown to induce apoptosis in human macrophages and monocytes, suggesting a role for these PPARs in the resolution of inflammation (17).

Most of the natural ligands of PPARs are derived from arachidonic acid metabolism through the action of cyclooxygenase or lipooxygenase and influence several inflammatory signaling pathways independently of PPAR ligation. In particular, cyclopentenone PGs have been reported to be lipids with the ability to react with key cysteine residues in proteins via the formation of Michael adducts (18), which might result in the modification of their biological activity (19, 20). Indeed, most of the effects observed with potential physiological PPAR agonists (i.e. PGs) cannot be obtained with pharmacological synthetic ligands, such as the TZDs. In view of the preceding results, we decided to investigate whether synthetic ligands of PPARs with a chemical structure distinct from that of TZDs might exert actions independently of the recruitment of these transcription factors. The macrophage cell line RAW 264.7 is a likely candidate to carry out these studies because these cells express very low levels of PPAR-, and the action of cyclopentenone PGs has been well established in these cells (19, 21, 22). We have used the TZD-unrelated PPAR synthetic agonists L-796,449 (3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic acid; referred to henceforth as compound G) and L-165,041 (4-[3-[2-propyl-3-hydroxy-4-acetyl]phenoxy]propyloxyphenoxyacetic acid; referred to henceforth as compound P) and compared their effects on the early signaling steps of macrophage activation with those of the TZD rosiglitazone and the cyclopentenone 15dPGJ₂. The binding affinities of compounds G and P, as well as those of PGs and rosiglitazone, have been measured in vitro with recombinant PPARs and in vivo using transactivation assays with reporter genes (23, 24). Compound G exhibits a high affinity for PPAR- and PPAR- (apparent K_d = 2 and 20 nM, respectively), and rosiglitazone has a high affinity for PPAR- (K_d  100 nM), whereas compound P is a poor agonist of both receptors (apparent K_d = 10 and 1 µM, respectively) (23). Compounds G and P exhibit structural homology in some groups of the molecule (23, 25), and their biological activities as antilipidemic and anti-inflammatory agents have been assayed previously (26). Our data show that compound G, but not rosiglitazone, inhibits the early steps of LPS signaling leading to NF-B activation well above the K_d for PPAR binding and enhances the synthesis of reactive oxygen and nitrogen intermediates promoting apoptotic cell death.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals-- Reagents were from Sigma, Roche, and Merck. The PPAR ligands compound G (L-796,449) and compound P (L-165,041) were from Merck Research Laboratories (Rahway, NJ; Fig. 1A). Antibodies and GST fusion proteins were from Santa Cruz Biotechnology (Santa Cruz, CA). PGs were from Calbiochem. Anti-(P)S³²IB Ab was from New England Biolabs (Beverly, MA). LPS was from Salmonella typhimurium (Sigma). Serum and media were from Biowhittaker (Walkersville, MD).

Cell Culture-- RAW 264.7 cells were seeded at 6-8 × 10⁴ cells/cm² in RPMI 1640 medium containing 2 mM glutamine, 10% fetal calf serum, and 50 µg/ml penicillin, streptomycin, and gentamicin. After 2 days in culture, the medium was replaced by phenol-red free RPMI 1640 medium supplemented with 0.5 mM arginine and 1% fetal calf serum, followed by the addition of the indicated stimuli. PGs, compound G and P ligands, and rosiglitazone were added 5 min before activation with LPS and IFN- (21).

Description of Plasmids-- The following plasmids were used: a 1-kilobase fragment corresponding to the 5'-flanking region of the NOS-2 gene and containing two B motifs was fused to a CAT reporter (pNOS-2(+,+).CAT vector). The pNOS-2(,).CAT vector contained mutated B sequences corresponding to nucleotides 971 to 961 and 85 to 75. The (B)₃ConA.CAT plasmid, which contains three copies of the B motif from the human immunodeficiency virus long terminal repeat enhancer linked to the minimal conalbumin A promoter, was used to measure B activity (21, 27). The ConA.CAT vector lacking the B tandem was used as a control and was not modulated by the ligands tested. The kSV₂.CAT plasmid was used as a reference for the efficiency of transfection. Plasmids were purified using EndoFree Qiagen columns (Hilden, Germany).

Transfection of RAW 264.7 Cells and Assay of CAT Activity-- Subconfluent cell cultures were washed twice with phosphate-buffered saline and maintained with 1.5 ml of RPMI 1640 medium and 1% fetal calf serum in 6-cm-diameter dishes. Cells were transfected for 6 h with FuGENE following the instructions of the supplier (Roche) and incubated overnight with 2 ml of RPMI 1640 medium plus 1% fetal calf serum before stimulation. Equal amounts of DNA were used for the transfection experiments. CAT activity was determined after 18 h of stimulation with the indicated factors, following a previous protocol based on TLC separation of acetylated [¹⁴C]chloramphenicol. The amount of acetylated substrate was quantified in a FUJI BAS1000 radioactivity detection system.

Expression and Purification of GST Fusion Proteins-- GST-IB-(1-54) wild type and the corresponding protein mutated in Ser^32/36 to Ala^32/36 were expressed in DH5F' Escherichia coli and purified by glutathione-Sepharose 4B chromatography (Amersham Pharmacia Biotech). Alternatively, purified GST-IB-(1-317) was obtained from Santa Cruz Biotechnology.

Analysis of RNA by Northern Blot-- Total RNA (2-4 × 10⁶ cells) was extracted using the guanidinium thiocyanate method, followed by electrophoresis in a 0.9% agarose gel containing 2% formaldehyde (28). The RNA was transferred to a Nytran membrane (NY 13-N; Schleicher & Schuell), and the levels of NOS-2 mRNA were determined using the EcoRI-HindII fragment from the NOS-2 cDNA, which was labeled with [-³²P]dCTP (Rediprime labeling kit; Amersham Pharmacia Biotech). The membranes were exposed to x-ray films (Hyperfilm, Amersham Pharmacia Biotech), and the intensity of the bands was measured by laser densitometry (Molecular Dynamics). The lane charge was normalized by hybridization with a ribosomal 18 S probe.

Preparation of Cytosolic and Nuclear Extracts-- Cells (3 × 10⁶) were washed with ice-cold phosphate-buffered saline, scraped off the dishes, and collected by centrifugation. Cell pellets were homogenized with 100 µl of buffer A (10 mM Hepes, pH 7.9, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mM NaF, 1 mM NaVO₄, and 10 mM Na₂MoO₄). After 10 min at 4 °C, Nonidet P-40 was added (0.5%, v/v), and the tubes were vortexed (15 s) and centrifuged at 8000 × g for 15 min. The supernatants were stored at 80 °C (soluble extracts), and the pellets were resuspended in 50 µl of buffer A supplemented with 20% glycerol and 0.4 M KCl and shaken for 30 min at 4 °C. After centrifugation for 15 min at 13,000 × g, the supernatants (nuclear protein extracts) were stored at 80 °C (29). Protein was determined using the Bio-Rad protein assay. Fractionation steps were carried out at 4 °C.

Electrophoretic Mobility Shift Assays (EMSAs)-- The oligonucleotide sequence 5'-TGCTAGGGGG ATTTTCCCTCTCTCTGT-3' corresponding to the consensus B site (nucleotides 978 to 952) of the murine NOS-2 promoter was annealed with the complementary DNA and end-labeled with Klenow enzyme in the presence of 50 µCi of [-³²P]dCTP (30, 31). The DNA probe (5 × 10⁴ dpm) was incubated for 15 min at 4 °C with nuclear protein extracts (3 µg) and 2 µg of polydeoxyguanylic-polydeoxy cytidylic acid, 5% glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, and 10 mM Tris-HCl, pH 7.8, in a final volume of 20 µl. The DNA-protein complexes were separated on native 6% polyacrylamide gels in 0.5% Tris borate-EDTA buffer. Supershift assays were carried out after incubation for 1 h at 4 °C of the nuclear extracts with 2 µg of Ab (anti-p50, anti-c-Rel, and anti-p65), followed by EMSA (data not shown). Normalization for lane charge was accomplished using PPAR- as probe (21). When the effect of 15dPGJ₂, compound G, compound P, and rosiglitazone on the binding of Rel proteins to the B motif was assayed in vitro, nuclear extracts from LPS/IFN--activated cells were treated for 5 min with these ligands before the addition of the probe.

Characterization of Proteins by Western Blot-- Cytosolic protein extracts were size-separated by 10% SDS-polyacrylamide gel electrophoresis. The gels were blotted onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) and incubated with anti-NOS-2, anti-IB, anti-IB, and anti-IKK2 Abs (Santa Cruz Laboratories, Santa Cruz, CA). In experiments using anti-(P)S³²IB Ab, the blot incubation solution contained 50 ng/ml GST-IB-(1-317) treated previously with alkaline phosphatase-agarose (21). The blots were submitted to sequential reprobing with Abs after treatment with 100 mM -mercaptoethanol and 2% SDS in Tris-buffered saline and heated at 60 °C for 30 min. The blots were revealed by ECL following the manufacturer's instructions (Amersham Pharmacia Biotech). Different exposure times of the films were used to ensure that bands were not saturated. Quantification of the films was performed by laser densitometry (Molecular Dynamics).

Determination of NO Synthesis-- NO release was determined spectrophotometrically by the accumulation of nitrite in the medium (phenol red-free). Nitrite was determined with Griess reagent by adding 1 mM sulfanilic acid, 100 mM HCl, and 1 mM naphthylenediamine (final concentrations). The absorbance at 548 nm was compared with a standard of NaNO₂. Results were expressed as the amount of nitrite released per milligram of cell protein.

Confocal Microscopy-- RAW 264.7 cells were grown on coverslips and incubated for 45 min with the indicated stimuli. After washing the covers twice with phosphate-buffered saline, the cells were fixed for 2 min with methanol at 20 °C, blocked for 20 min with 3% bovine serum albumin at room temperature, and incubated for 30 min with 1:100 anti-IB or anti-IB Abs. After three washes with ice-cold phosphate-buffered saline, the level of IB proteins was determined using a secondary Ab (1:300) against rabbit IgG conjugated with Cy3 (Amersham Pharmacia Biotech). Cells were visualized on a MRC-1024 confocal microscope (Bio-Rad), and the fluorescence was measured and electronically evaluated. Laser sharp software (Bio-Rad) was used to determine the relative intensity of the fluorescence per pixel and the percentage of cytosolic and nuclear localization.

Measurement of IKK2 Activity-- Cells (10⁷) were homogenized in buffer A and centrifuged for 10 min in a microcentrifuge. The supernatant (1 ml) was precleared, and IKK2 was immunoprecipitated with 1 µg of anti-IKK2 Ab (New England Biolabs). After extensive washing of the immunoprecipitate with buffer A, the pellet was resuspended in kinase buffer (20 mM Hepes, pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mM NaF, 1 mM NaVO₄, 10 mM Na₂MoO₄, and 10 nM okadaic acid). The kinase activity was assayed in 100 µl of kinase buffer containing 100 ng of immunoprecipitation protein and 50 µM [-³²P]ATP (0.5 µCi), using as substrate 100 ng of GST-IB-(1-317) or GST-IB-(1-54) and the corresponding Ala^32/36 mutated protein. Aliquots of reaction were stopped at various times in 1 ml of ice-cold buffer A supplemented with 5 mM EDTA. The same protocol was used when the activity of IKK2 was followed by Western blot using anti-(P)S³²IB Ab, except that 1 mM MgATP was used instead of [-³²P]ATP. GST-IB was purified by glutathione-Sepharose 4B chromatography and analyzed by 10% SDS-polyacrylamide gel electrophoresis. The linearity of the kinase reaction was confirmed over a period of 30 min (21).

Flow Cytometric Analysis of m-- Cells were incubated for 15 min at 37 °C in the presence of the potential-sensitive probe CMXRos (40 nM), followed by analysis in a FACScan flow cytometer. The fluorescence in the presence of uncoupling agent m-chlorophenylhydrazone carbonylcyanide (10 µM) was considered to be 100%, and values were calculated as a percentage of the change of fluorochrome fluorescence (32, 33).

Measurement of ROI Synthesis-- Cells challenged for 8 h with different stimuli were incubated for 15 min with 10 µM DCFH or HE, and the fluorescence corresponding to the oxidized probes was followed by analysis in a flow cytometer as described previously (32, 34). Incubation of cells with 50 µM t-butyl hydroperoxide and 20 µM peroxynitrite was used as positive control of ROI and reactive nitrogen intermediate release.

Analysis of Apoptosis-- Flow cytometric measurement of propidium iodide staining was performed after incubation of the cells with 0.005% propidium iodide, following a previous protocol (35, 36). Cells were analyzed in a FACScan cytometer (Becton & Dickinson, San Jose, CA) equipped with a 25-mW argon laser. The quantification of the percentage of apoptotic cells was calculated using a dot plot of the forward scatter against the propidium iodide fluorescence. Cell sorting and analysis of viable and apoptotic populations was performed to confirm the criteria of gating (37). Activation of caspase 3 (and caspase 7) was determined measuring the DEVDase activity in the cytosolic extracts, following the appearance of fluorescence from N-acetyl-DEVD-7-amino-4-methylcoumarin as substrate. The corresponding peptide aldehyde and Z-VAD.fmk were used to inhibit caspase activity and to ensure the specificity of the reaction. The linearity of the caspase assay was determined over a 20-min reaction period.

Data Analysis-- The number of experiments analyzed is indicated in each figure. Statistical differences (p < 0.05) between mean values were determined by one-way analysis of the variance followed by Student's t test. In experiments using x-ray films (Hyperfilm), different exposure times were used to avoid saturation of the bands.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Compound G and, to a Lesser Extent, Compound P Inhibit NO Synthesis in RAW Cells-- Incubation of RAW 264.7 cells with the PPAR ligands compound G, compound P, 15dPGJ₂, and rosiglitazone did not promote the synthesis of NO. However, when cells were activated with LPS and IFN-, the presence of compound G, compound P, and 15dPGJ₂, but not rosiglitazone, significantly inhibited the accumulation of NO in the culture medium through a mechanism that involved a reduction in the levels of NOS-2 mRNA and protein (Fig. 1, B and C). The apparent K_i values for NO synthesis were 1.1, 6, and 0.6 µM for compound G, compound P, and 15dPGJ₂, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Inhibition of NOS-2 expression and NO synthesis by PPAR agonists. RAW 264.7 cells were incubated with the indicated concentrations of compound G, compound P, 15dPGJ2, and rosiglitazone (see the chemical structures in A), and 5 min later, the macrophages were activated with 200 ng/ml LPS and 20 units/ml IFN-. The release of nitrite to the medium was measured after 18 h (100% = 48 nmol NO2 + NO3/mg protein; B). The levels of NOS-2 and -actin protein (18 h) and RNA (4 h) were measured in activated cells treated with 2 µM 15dPGJ2 and 5 µM compound G, compound P, and rosiglitazone (C). Normalization of RNA lane charge was carried out with 18 S ribosomal RNA. Results show the mean of three experiments (left) and one of four representative experiments (right).

Compound G and 15dPGJ₂ Inhibit NF-B Activation-- The low level of NOS-2 mRNA measured in cells treated for 4 h with compound G suggests that this molecule interferes with the stimulatory signaling elicited after LPS/IFN- challenge. Because NF-B activation has been reported as a necessary event for NOS-2 expression (31, 38), the binding of NF-B proteins to the B motif of the NOS-2 promoter was assayed by EMSA. As Fig. 2 shows, the binding of the dimers p50.p50 and p50.p65 to the B sequence was impaired in cells incubated with 15dPGJ₂ and compound G. However, treatment with compound P or rosiglitazone did not affect NF-B binding. When the protein levels of cytosolic IB were determined after 30 min of challenge, an inverse correlation was observed with respect to the corresponding B activity. The subcellular distribution of IB and IB in activated RAW 264.7 cells was further analyzed by confocal microscopy, revealing an impaired degradation after incubation with 15dPGJ₂ and compound G (Fig. 3). These results suggest that compound G inhibits early signaling after LPS and IFN- challenge (in particular, the activation of NF-B). To investigate whether these PPAR ligands can affect the transcription of genes requiring NF-B activity, RAW 264.7 cells were transfected with CAT reporter plasmids containing the B sites of the murine NOS-2 promoter or a 3× tandem of B motifs linked to the minimal promoter of the conalbumin A gene. After stimulation with LPS/IFN- for 18 h, CAT activity was measured. As Fig. 4 shows, 15dPGJ₂ and compound G inhibited CAT expression, whereas this effect was not observed in cells treated with rosiglitazone. The moderate inhibitory effect of compound P was more important on the pNOS.CAT reporter than on the (B)₃ConA.CAT counterpart, suggesting an action on NOS-2 transcription distinct from NF-B activation.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of PPAR agonists on NF-B activity. RAW 264.7 cells were treated with 2 µM 15dPGJ2 and 5 µM compound G, compound P, and rosiglitazone and activated for 30 min with LPS/IFN-. Cells were homogenized, and the NF-B activity was determined in nuclear extracts by EMSA. Supershift assays (data not shown) indicated that the upper and lower bands were composed of p50.p65 and p50.p50 dimers, respectively. The binding of nuclear extracts to a PPAR- motif was used to ensure a constant level of proteins in the lanes. The levels of IB in the cytosol were determined by Western blot. Results are from one of three representative experiments. A densitometric analysis of the p50.p65 and IB band intensities (mean + S.D.) is shown in the right panel.

View larger version (68K): [in this window] [in a new window]   Fig. 3.   Immunocytochemical analysis of IB levels in RAW 264.7 cells treated with PPAR agonists. Cells were treated for 45 min with the indicated stimuli as described in the Fig. 2 legend. After fixation, the cells were incubated with anti-IB and anti-IB Abs and stained with Cy3-labeled anti-rabbit IgG Ab (red). The distribution of fluorescence was analyzed by confocal microscopy, and the average cell fluorescence (n = 50-70; mean + S.D.) was quantified.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effect of PPAR agonists on the expression of CAT activity in RAW 264.7 cells transfected with the pNOS-2.CAT and (B)3ConA.CAT reporter plasmids. Cells were transfected with the indicated plasmids with FuGENE, followed by treatment with 2 µM 15dPGJ2, 5 µM compound G and compound P, and 10 µM rosiglitazone. After activation with LPS/IFN-, the CAT activity was measured at 18 h. A kSV2.CAT plasmid was used to normalize the efficiency of the transfection and to discard nonspecific effects of the ligands. Results are the mean + S.D. of three experiments assayed per duplicate. * and **, p < 0.01 and p < 0.005 with respect to the corresponding LPS/IFN- condition, respectively.

IKK2 Activity Was Inhibited by Compound G and 15dPGJ₂-- NF-B activity is dependent on IKK2 activation (21, 39, 40). To investigate the effect of the PPAR agonists on IKK2, the kinase was immunoprecipitated from LPS/IFN--activated cells, and the activity was measured in vitro. As Fig. 5 shows, the incorporation of [³²P]phosphate into GST-IB was impaired in cells treated with compound G and 15dPGJ₂, whereas compound P and rosiglitazone did not exert a significant inhibition on this activity. This phosphorylation involved Ser³² as deduced by Western blot using a specific anti-(P)S³²IB Ab. Moreover, using a GST-A^32/36-IB peptide, we confirmed that the corresponding serine residues were the phosphorylation sites in the peptide fragment assayed (amino acids 1-54). To evaluate the ability of these molecules to inhibit IKK2 activity in vitro, cells were activated with LPS/IFN-, and the kinase was immunoprecipitated. As Fig. 6A shows, only compound G and 15dPGJ₂ significantly inhibited the kinase activity. Following this in vitro approach, the NF-B activity of nuclear extracts incubated in vitro with 15dPGJ₂, compound G, compound P, and rosiglitazone was assayed. As Fig. 6B shows, only 15dPGJ₂ abrogated the binding activity.

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Inhibition of IKK2 activity. Cells were treated for 15 min with the indicated stimuli at the concentrations indicated in the Fig. 2 legend, and IKK2 was immunoprecipitated. The kinase activity was determined in vitro by incorporation of [32P]phosphate into GST-IB or by Western blot using a specific anti-phospho-S32IB Ab. Results are from one of three representative experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   In vitro effect of PPAR agonists on IKK2 and NF-B activity. IKK activity was immunoprecipitated from LPS/IFN--activated cells (15 min), and the effect of 15dPGJ2, compound G, compound P, and rosiglitazone on IKK2 was evaluated in vitro by measuring the phosphorylation of GST-IB-(1-317) (A). Aliquots of nuclear proteins from cells activated for 30 min with LPS/IFN- were incubated for 5 min with the indicated concentrations of PPAR agonists, followed by EMSA analysis of the binding to the B motif of NOS-2 (B). Results show the dose-dependent effect of two preparations of IKK assayed per triplicate (mean + S.D.) and the mobility shift of one of three representative experiments.

Compound G and 15dPGJ₂ Increase the Synthesis of ROI and Promote Apoptosis-- The aforementioned results suggest an impairment of early inflammatory signaling in cells treated with compound G and 15dPGJ₂. However, under these conditions, we observed morphological changes compatible with an enhanced apoptotic rate, despite the attenuated synthesis of NO. Therefore, we investigated the effect of the PPAR ligands on the induction of oxidative stress in activated macrophages. Incubation of cells with these PPAR agonists did not affect ROI synthesis as determined by the oxidation of DCFH and HE (Fig. 7). However, in LPS/IFN--activated cells 15dPGJ₂ and compound G significantly enhanced the oxidation of DCFH but failed to affect the oxidation of HE. Interestingly, in the absence of NO synthesis accomplished after inhibition of NOS-2 with 1400W (N-(3-aminomethylbenzyl)acetamidine), the oxidation of DCFH was drastically reduced, whereas that of HE increased in cells treated with 15dPGJ₂ and compound G. These results suggest that these compounds increase the synthesis of reactive oxygen and nitrogen intermediates in macrophages (see "Discussion") as described previously (37). The effect of the enhanced synthesis of reactive species on the mitochondrial permeability transition pore was evaluated using the potential-sensitive probe CMXRos. As Fig. 8A shows, the fluorescence observed in LPS/IFN--treated cells increased over the value of control cells, data that are in agreement with the findings of previous reports (32, 33). Moreover, the increase in CMXRos fluorescence can be abolished after a short incubation with m-chlorophenylhydrazone carbonylcyanide, which dissipates the mitochondrial inner membrane potential. Because the synthesis of ROI and reactive nitrogen intermediate promotes apoptosis in macrophages, and the increase of CMXRos fluorescence is a characteristic feature of mitochondrial-dependent apoptotic triggering in these cells (37), we determined the extent of apoptosis in RAW 264.7 cells treated with these PPAR ligands. As Fig. 8B shows, 15dPGJ₂ and compound G promoted apoptosis in activated cells, whereas compound P exerted a moderate apoptotic effect, and rosiglitazone required concentrations higher than 10 µM. This induction of apoptosis was accompanied by an increase in DEVDase activity, reflecting the activation of caspase 3/caspase 7 (Fig. 8C).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Synthesis of reactive species by PPAR ligands. Cells were treated for 8 h with the indicated stimuli in the absence or presence of the NOS-2 inhibitor 1400W (50 µM). The oxidation of DCFH and HE was determined by flow cytometry. Results show the mean + S.D. of three experiments. *, p < 0.005 with respect to the corresponding control.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Changes of CMXRos fluorescence and induction of apoptosis in macrophages treated with PPAR ligands. Cells were treated for 8 h with the indicated stimuli, and the accumulation of CMXRos fluorescence was determined by flow cytometry. Cells treated for 10 min with the uncoupling agent m-chlorophenylhydrazone carbonylcyanide were used to evaluate the minimal fluorescence per cell (A). The percentage of apoptotic cells was determined at 18 h by flow cytometry after staining cells with propidium iodide (B). The DEVDase activity of cell extracts treated for 8 h with the indicated stimuli was assayed fluorometrically (C). Results show the mean (+ S.D.) of three experiments. *, p < 0.05 with respect to the LPS/IFN- condition.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Controversy exists in the literature with regard to the relative contribution of some PPAR- agonists to the regulation of transcription through nuclear receptor ligation-dependent (13-16) and -independent mechanisms (19, 21, 22). PPAR- activation in macrophages impairs the expression of genes requiring activator protein-1, NF-B, and signal transducer and activator of transcription-1, resulting in the abrogation of the transcription of NOS-2, gelatinase B, and other genes related to inflammation (14). Similar results were observed in human monocytes (13). However, other groups observed that prostanoids released after the expression of cyclooxygenase-2, in particular, 15dPGJ₂, exerted rapid and early effects on the activation of NF-B that were independent of PPAR- engagement and due to the formation of Michael's adducts between the cyclopentenone moiety of the prostaglandin and functional cysteine residues in target proteins (18). Interestingly, these effects could not be reproduced after treatment of the cells with TZDs. Moreover, the possibility of a direct interaction of the cyclopentenone with key cysteine residues in the PPAR- exists, and the diversity in the structure of its agonists might be explained in this way.

The results reported in this work indicate that the high-affinity PPAR- ligand compound G exerts effects on early LPS-dependent signaling pathways, which are independent of the transcriptional activity of this nuclear receptor. In RAW 264.7 cells, the protein levels of PPAR- determined by Western blotting nuclear extracts were negligible when compared with those prevailing in elicited peritoneal macrophages (14, 37). Moreover, Northern blot analysis of the mRNA expressed in these cells showed minimal levels of PPAR-1 and undetectable amounts of PPAR-2 (data not shown), data that were in agreement with those published previously (26). Therefore, it is likely that most of the effects of compound G in RAW 264.7 cells are independent of nuclear receptor engagement. Indeed, when experiments on NOS-2 expression and NO synthesis similar to those described in Fig. 1 were performed in elicited peritoneal macrophages (which contain large amounts of PPAR-), a notable increase in the inhibitory capacity of compound G, compound P, and rosiglitazone was observed, reflecting the additional contribution of the transrepression mechanism dependent on PPAR- activation (16). Our data show that compound G inhibited IKK2 activation in vitro and in vivo, which resulted in the impairment of IB targeting in the cytosol and abrogation of NF-B activity, as described previously (15, 19, 40-42). Because IB is also expressed in RAW 264.7 cells (21), and it was proposed that this form contributed to sustained activation of NF-B (43), we analyzed the effect of compound G on the subcellular distribution of IB proteins in the course of LPS activation. An inhibition of the degradation of both IBs, together with an increase in the presence of these proteins in the nucleus, was observed, presumably contributing to the impairment of NF-B activity (44). However, we were unable to observe direct effects of compound G on NF-B binding activity in vitro by measuring the interaction of the p50-p65 complexes with the B motif of NOS-2. This was not the case with 15dPGJ₂, which exhibited an inhibitory interaction with p65 in vitro, probably via formation of a Michael adduct (20). Moreover, studies on the effect of compound G on purified proteasome showed a significant inhibition of the basal activity assayed with N-succinyl-Leu-Leu-Val-Tyr as substrate (data not shown), suggesting that targeting of IB by the proteasome was also affected.

Most of the results obtained with compound G on early LPS signaling showed a pattern similar to that reported for 15dPGJ₂ (21), although this molecule does not contain a cyclopentenone motif. However, previous work on the mechanism of activation of PPAR- by L-764,406 identified the covalent modification of Cys³¹³ located in helix 3 of the nuclear receptor by a chlorine residue of the agonist as a requirement for the activation process (23, 25). Interestingly, both compound G and L-764,406 are non-TZD agonists that share similar structures around the chlorine residues. With these data in mind, it is possible that compound G interacts with the cysteine residues of IKK2. Accordingly, IKK2 activity was inhibited by compound G both in vivo and in vitro, although we cannot precisely determine the mechanism of action. Moreover, the effects of compound G on LPS signaling exhibited some degree of specificity because, for example, the activation of c-Jun N-terminal kinase, which has been well documented in macrophages after LPS challenge (45, 46), was not affected by compound G at doses up to 10 µM (data not shown).

In a previous report using RAW 264.7 cells and human monocytes stimulated with a very low concentration of LPS from Salmonella minnesotta (0.1 ng/ml), pretreatment for 1 h with compound G and compound P did not affect the secretion of tumor necrosis factor  and interleukin 6 as markers of cell activation, whereas 15dPGJ₂ inhibited the synthesis of these pro-inflammatory cytokines (26). From these results, it was concluded that compound G and compound P lacked anti-inflammatory activity in vivo. However, the activation of NF-B elicited by 0.1 ng/ml LPS was barely detectable in the RAW 264.7 cells used in this work, and a concerted action of LPS and IFN- was required to express NOS-2 and to mediate a significant synthesis of NO (37). In this regard, it is worth mentioning that 15dPGJ₂ is more efficient than TZDs in terms of inhibition of cytokine production by human monocytes, suggesting that, in addition to PPAR- activation, cyclopentenone prostaglandins act on additional targets that exert an important control on the inflammatory process (13). In line with this, it has been shown in macrophages that oxidized low density lipoproteins inhibit the LPS-dependent production of interleukin 12, a cytokine that potently induces the synthesis of IFN- and T-cell activation, through a dual mechanism that involves both the inhibition of the recognition between the NF-B complex and the DNA B sites and the physical interaction between NF-B and PPAR- (47).

In addition to the inhibition of IKK2 activity, it has been shown that compound G (but not rosiglitazone) increases the synthesis of reactive oxygen species in activated RAW 264.7 cells, and this process contributes to the induction of apoptosis. Indeed, treatment of LPS/IFN--activated cells with 15dPGJ₂ triggers apoptosis via an increase in the production of superoxide that results in the synthesis of significant amounts of peroxynitrite (37).

The contribution to apoptosis of PPAR- agonists has been described in various cell types; activation of PPAR- with thiazolidinediones (49653; Life Technologies, Inc.) leads to apoptosis of activated human macrophages (17, 48). However, as noted by Chinetti et al. (17), the induction of apoptosis in human macrophages by TZD was observed preferentially in activated cells and at much higher concentrations (2-3 orders of magnitude) than those required for PPAR- activation. Also, 15dPGJ₂ and TZD potently induced caspase activation in human endothelial cells, both in human umbilical vascular endothelial cells and immortalized endothelial cells (49). In the same vein, 15dPGJ₂ and troglitazone induced apoptosis in synoviocytes from patients with rheumatoid arthritis, which contributed to the amelioration of the inflammatory process (50), and oxidized low density lipoprotein, which caused endothelial cell apoptosis in part via caspase activation and also through enhancement of the synthesis of reactive oxygen species (51). In RAW 264.7 cells 15dPGJ₂ and compound G, but not rosiglitazone, triggered apoptosis, suggesting that this process is mainly accomplished through PPAR--independent mechanisms.

The ability of NF-B to sense oxidative stress and to integrate this signaling in terms of regulation of cell viability (induction of apoptosis and necrosis) has been a subject of current debate (52). Because the main regulatory step controlling NF-B activity is located at the IKK level (39, 40), it might be suggested that non-TZD PPAR- agonists, such as 15dPGJ₂ and compound G, which inhibit IKK activity, offer the possibility to control NF-B activity at different points. This is important for the resolution of inflammatory processes in which not only the synthesis of cytokines and chemical mediators (NO and reactive oxygen species) needs to be impaired, but the removal of activated cells by an efficient apoptotic mechanism, reinforced by a deficient NF-B activation, is also required (53, 54).

	ACKNOWLEDGEMENTS

We thank E. Lunidn, Dr. M. A. Moro, and Dr. B. de las Heras for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Grants PM98-0120 and FD97-1432 from the Comisión Interministerial de Ciencia y Tecnología, and Sociedad Española de Nefrología, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed. Fax: 3491-544-7254/3491-394-1782; E-mail: boscal@eucmax.sim.ucm.es

Published, JBC Papers in Press, July 3, 2001, DOI 10.1074/jbc.M102472200

² A. Castrillo, M. Mojena, S. Hortelano, and L. Boscá, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; CMXRos, chloromethyl X-rosamine; 15dPGJ₂, 15-deoxy-^12,14-prostaglandin J₂; DCFH, 2',7'-dichlorofluorescein diacetate; HE, hydroethidine; IKK, IB kinase; IFN, interferon; NOS-2, NO synthase-2; NF-B, nuclear factor B; PG, prostaglandin; TZD, thiazolidinedione; GST, glutathione S- transferase; Ab, antibody; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; EMSA, electrophoretic mobility shift assay; ROI, reactive oxygen intermediate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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