The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen1,2,3. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates3,4,5,6. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes7,8,9,10. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages11, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.
To determine whether 15d-PGJ2 might be involved in the regulation of macrophage gene expression by activating PPAR-γ, we performed RNase protection assays using RNA derived from different macrophage populations. Resting bone-marrow-derived macrophages express low levels of PPAR-γ mRNA, whereas activated peritoneal macrophages express high levels of PPAR-γ (Fig. 1a). Peritoneal macrophages treated with 15d-PGJ2 exhibited morphological features typical of resting cells even in the presence of interferon-γ (IFN-γ), which is a potent inducer of macrophage activation (Fig. 1b). Similar effects were observed in the presence of BRL 49653 (Fig. 1b), which is a specific PPAR-γ agonist of the thiazolidinedione family5 (Fig. 1b). These observations suggest that PPAR-γ might inhibit the expression of genes that become upregulated during macrophage differentiation and activation. To evaluate this possibility, we examined the effects of 15d-PGJ2 and other PPAR-γ ligands on expression of the inducible nitric oxide synthase (iNOS), which exerts cytotoxic effects on invading microorganisms and is upregulated in activated macrophages in response to IFN-γ12. Treatment of peritoneal macrophages with increasing concentrations of 15d-PGJ2 or synthetic PPAR-γ ligands led to a dose-dependent inhibition of IFN-γ-dependent nitrite production (Fig. 1c) and inhibited the induction of iNOS mRNA (Fig. 1d). Although highly specific for PPAR-γ5,6,9,13, BRL 49653, troglitazone and GW 2090 were less potent than the natural ligand 15d-PGJ2 as inhibitors of iNOS activity.
As a second marker of macrophage gene expression, we examined effects of 15d-PGJ2 on expression of gelatinase B, a matrix metalloproteinase that is upregulated during macrophage activation and contributes to tissue damage in acute and chronic inflammation14,15. Treatment of bone-marrow-derived macrophages that express low levels of PPAR-γ with 15d-PGJ2 had little effect on gelatinase B mRNA levels (Fig. 1e). In contrast, 15d-PGJ2 treatment of peritoneal macrophages that express high levels of PPAR-γ resulted in a near-complete inhibition of gelatinase B mRNA expression (Fig. 1e). To further investigate the mechanisms of inhibition, the effects of 15d-PGJ2 on regulation of the gelatinase B promoter were studied in U937 histiocytic leukaemia cells. These cells express PPAR-γ16, and can be induced to differentiate into macrophage-like cells by treatment with the phorbol ester TPA. When U937 cells were transfected with a gelatinase B promoter-luciferase reporter gene, TPA treatment resulted in a marked increase in promoter activity (Fig. 2a). This activity was strongly inhibited by concurrent treatment of the cells with 15d-PGJ2. Overexpression of PPAR-γ potentiated the inhibitory effects of 15d-PGJ2, suggesting that they were mediated by PPAR-γ (Fig. 2a). Transcriptional activation of the gelatinase B promoter is dependent on binding sites for AP-1 proteins present in proximal and distal regulatory regions17,18 (Fig. 2a). To determine whether other AP-1-dependent promoters are also subject to negative regulation by 15d-PGJ2, we examined its effects on transcriptional activation of the scavenger receptor A (SR-A) gene. The SR-A gene encodes a macrophage-specific cell-surface protein implicated in Ca2+-independent cell-adhesion events and the uptake of polyanionic macromolecules, including oxidized low-density lipoprotein19. Transcriptional activation of the SR-A promoter in response to TPA is dependent on combinatorial interactions between AP-1 and Ets transcription factors20. Similar to the results observed for gelatinase B, 15d-PGJ2 inhibited TPA-dependent activation of the SR-A promoter in a manner that was potentiated by overexpression of PPAR-γ (Fig. 2b).
To examine the mechanisms by which 15d-PGJ2 inhibits iNOS expression, we investigated regulation of the iNOS promoter in RAW 264.7 macrophages. As well as having binding sites for AP-1 proteins, the iNOS promoter also contains multiple binding sites for Stat1 and NF-κB (Fig. 2d), which are required for maximal responses to IFN-γ and lipopolysaccharide (LPS), respectively21,22. RAW 264.7 cells express very low levels of PPAR-γ mRNA (data not shown), such that activation of a PPAR-dependent promoter (AOx-TK) required transfection of a PPAR-γ expression plasmid (Fig. 2c). This cell line therefore allowed a direct assessment of the role of PPAR-γ in mediating the inhibitory effects of 15d-PGJ2 and synthetic PPAR-γ ligands on macrophage gene expression. In the absence of a co-transfected PPAR-γ expression plasmid, treatment of RAW 264.7 macrophages with 15d-PGJ2 at concentrations up to 1 µM had little or no effect on iNOS activation by IFN-γ (Fig. 2d). However, when a PPAR-γ expression plasmid was transfected into these cells, 15d-PGJ2 strongly inhibited IFNγ-dependent activation of the iNOS promoter (Fig. 2d), as well as the synergistic transcriptional response to the combination of IFN-γ and LPS (Fig. 3a). Thus 15d-PGJ2 inhibits induction of the iNOS promoter by a PPARγ-dependent mechanism. The inhibitory effects of 15d-PGJ2 and PPAR-γ were promoter specific, because no effect was observed on the expression of several other viral and cellular promoters, including the β-actin, HSV thymidine kinase, RSV and CMV promoters (data not shown).
Inhibition of IFN-γ-dependent induction of iNOS promoter activity in RAW 264.7 cells was observed for the 15d-PGJ2 precursor, PGD2, but not for other prostaglandin metabolites, including PGE2 and PGH2 (Fig. 3b). Because PGD2 does not bind to PPAR-γ7,8, and no inhibitory activity was observed in the absence of co-transfected PPAR-γ (data not shown), these results indicate metabolic conversion of PGD2 to an activating ligand by RAW 264.7 macrophages. PPAR-γ-dependent inhibition of the iNOS promoter was also observed for several synthetic ligands (Fig. 3c), including the thiazolidinedione BRL 49653, as well as the fibric-acid derivatives GW9820, GW2090 and GW2334 (Fig. 3c).
Because activation of the SR-A, gelatinase B and iNOS promoters depends on combinatorial interactions between members of the AP-1, STAT and NF-κB families of transcription factors, we evaluated minimal promoters containing binding sites for these factors to determine whether they might be targets for negative regulation by 15d-PGJ2 and synthetic PPAR-γ ligands. We found that 15d-PGJ2 effectively inhibited each of these promoters in a PPARγ-dependent manner (Fig. 4), but did not inhibit a promoter containing binding sites for SP-1 (data not shown). Thus activation of PPAR-γ by 15d-PGJ2 results in inhibitory effects on at least three different classes of transcription factors that play general roles in regulating inflammatory responses in many cell types, including macrophages. The synthetic PPAR-γ ligand BRL 49653 also inhibited STAT, NF-κB and AP-1 activities in a PPARγ-dependent manner, but did so less effectively than 15d-PGJ2, particularly in the case of the AP-1-dependent promoter. These results are consistent with the requirement for higher concentrations of BRL 49653 to inhibit iNOS expression in primary macrophages and RAW 264.7 cells transfected with PPAR-γ expression vectors.
Previous studies have demonstrated that prostaglandin D2 synthase, which is required for 15d-PGJ2 synthesis, is predominantly expressed in macrophages and specialized antigen-presenting cells11. The receptor PPAR-α, which is related to PPAR-γ, can be weakly activated by 15d-PGJ2, but PPAR-α is not expressed in activated macrophages (presented as Supplementary information). Although activated macrophages express both PPAR-γ (Fig. 1) and PPAR-δ (data not shown), 15d-PGJ2 is not an effective ligand for PPAR-δ10. These observations therefore raise the possibility that, in addition to its proposed roles in the regulation of adipocyte differentiation and glucose metabolism, PPAR-γ may also regulate inflammatory responses as a result of local production of 15d-PGJ2. Taken together with recent evidence that fatty acids are physiological ligands of PPAR-γ in adipose tissue9, our results suggest that PPAR-γ is regulated by distinct classes of ligands in different locations. PPAR-γ is under the transcriptional control of at least two distinct promoters3, suggesting that different signals regulate its expression in fat cells and macrophages. With regard to the different efficacies of natural and synthetic PPAR-γ ligands as inhibitors of macrophage gene expression, we note that dissociation of target gene activation and AP-1 inhibition has previously been described for ligands of the retinoic acid receptor23. Thiazolidinediones, 15d-PGJ2 and fibric acid derivatives represent three structurally distinct classes of activating ligands, and are thus likely to induce different receptor conformations that account for these observations. Taken together, these results suggest that synthetic PPAR-γ ligands that are analogues of 15d-PGJ2 may have therapeutic applications in diseases in which activated macrophages play prominent pathogenic roles, such as rheumatoid arthritis and atherosclerosis.
Cell culture. Murine bone-marrow progenitor cells and primary peritoneal macrophages were isolated and cultured as described24. Recombinant macrophage colony-stimulating factor (M-CSF) was used at 20 ng ml−1. Murine IFN-γ was used at 500 U ml−1. U-937 (ATCC) and THP-1 (ATCC) cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (Gemini), 100 U ml−1penicillin and 100 mg ml−1streptomycin. RAW 264.7 (ATCC) cells and HeLa (ATCC) cells were cultured under the same conditions except that Dulbecco's modified Eagle's medium (DMEM) was used.
RNA analysis. Total RNA was isolated by the guanidium thiocyanate method and RNase protection assays were performed as described25. The antisense probe for gelatinase B corresponded to nucleotides 1779–1999. The antisense PPAR-γ probe corresponded to nucleotides 800–1093. Northern blot analysis of iNOS mRNA used 10 µg of total RNA per treatment conditions. The iNOS probe consisted of a 500-base pair fragment of the cDNA corresponding to nucleotides 2849–3349. To control for equivalency of RNA loading and transfer, blots were hybridized with the cDNA for glyceraldehyde-3-phosphate dehydrogenase. Probes were labelled by random priming.
Nitrite concentrations. Supernatants from thioglycolate-elicited peritoneal macrophages cultured with or without IFN-γ in the presence or absence of 15d-PGJ2 or synthetic ligands were mixed with an equal volume of the Griess reagent in 96-well plates, gently shaken for 20 min at room temperature, and read in a microplate reader at 550 nm (ref. 26). Readings for experimental samples were read off a standard curve generated by dilutions of NaNO2. The assay is sensitive to nitrite concentrations of approximately 1 µM.
Transcription assays. U937 cells were transiently transfected with luciferase reporter genes by electroporation27 in 200 µl OptiMEM (Gibco) with 10 µg reporter plasmid and 0.1 µg β-actin-lacZ reporter plasmid, as internal transfection control, at 250 V, 960 µF using a Biorad electroporator with capacitance extender. RAW 267.4 cells were transfected using lipofectamin (Gibco-BRL). HeLa cells were transfected using Ca2+phosphate. Salmon sperm DNA or vector DNA was used to balance all transfection groups to equal amounts of transfected DNA. Luciferase and β-galactosidase enzymatic activities were determined20 and luciferase activity was normalized to the β-galactosidase standard. The reporter gene constructs for the SR-A gene, NOS, gelatinase B, AOx-TK and β-actin have been described18,20,22.
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We thank C. Archer for assistance with nitrate measurements, and T. Schneiderman for assistance with manuscript preparation. A.C.L. is supported by a Physician Scientist grant from the NIH. C.J.K. is a clinical investigator of the Medical Research Service, Department of Veterans Affairs. C.K.G. is an established investigator of the American Heart Association. These studies were supported by NIH grants to C.J.K. and C.K.G.
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