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The Cyclopentenone-Type Prostaglandin 15-Deoxy-^12,14-Prostaglandin J₂ Inhibits CD95 Ligand Gene Expression in T Lymphocytes: Interference with Promoter Activation Via Peroxisome Proliferator-Activated Receptor--Independent Mechanisms¹

Marco Cippitelli^2,*,, Cinzia Fionda^*,, Danilo Di Bona, Aldo Lupo, Mario Piccoli^*, Luigi Frati^3,*, and Angela Santoni^3,*,,

^* Dipartimento di Medicina Sperimentale e Patologia, Istituto Pasteur-Fondazione Cenci Bolognetti, University "La Sapienza," Rome, Italy; Regina Elena Cancer Institute, Rome, Italy; Istituto Mediterraneo di Neuroscienze "Neuromed," Pozzilli, Italy; and Dipartimento di Biopatologia e Metodologie Biomediche, University of Palermo, Palermo, Italy

	Abstract

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15-Deoxy-^12,14-PGJ₂ (15d-PGJ₂) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. 15d-PGJ₂ is a natural ligand of the peroxisome proliferator-activated receptor (PPAR)- nuclear receptor, but relevant PPAR-independent actions mediated by this prostanoid have been described. Fas (APO-1/CD95) and its ligand (Fas-L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death and has been implicated in diseases in which lymphocyte homeostasis is compromised. Moreover, several studies have described the pathogenic functions of Fas and Fas-L in vivo, particularly in the induction-progression of organ-specific autoimmune diseases. In this study we describe the effect of 15d-PGJ₂ on the activation of the fas-L gene in T lymphocytes. We show that 15d-PGJ₂ inhibits fas-L mRNA expression, activation-induced cell death, and fas-L promoter activity by mechanisms independent of PPAR and mediated by its chemically reactive cyclopentenone moiety. Our data indicate that 15d-PGJ₂ may repress fas-L activation by interfering with the expression and/or transcriptional activity of different transcription factors (early growth response types 3 and 1, NF-B, AP-1, c-Myc, Nur77) whose altered balancing and transactivation may contribute for overall repression of this gene. In addition, the activation/expression of the heat shock response genes HSF-1 and HSP70 is not directly involved in the repression, and the electrophilic molecule cyclopentenone (2-cyclopenten-1-one) may reproduce the effects mediated by 15d-PGJ₂. These results suggest that modulation of Fas-L by 15d-PGJ₂ in T cells may represent an additional tool to consider for treatment of specific autoimmune and inflammatory disorders.

	Introduction

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The prostanoid 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂)⁴ is a cyclopentenone-type PG (cyPG) of the J₂ series derived by dehydration of PGD₂ (a major cyclooxygenase product in a variety of tissues) produced by different cells, including those of the immune system such as mast cells, platelets, T cells, and macrophages (Refs. 1, 2, 3 and references cited therein).

In the past few years this prostanoid has been characterized as a natural ligand that activates the peroxisome proliferator-activated receptor (PPAR), a nuclear receptor originally implicated in the regulation of adipogenesis (4) and also expressed by a variety of immune cells, including macrophages, T cells, B cells, and dendritic cells (1, 3). Moreover, an increasing number of reports have also described different effects mediated by 15d-PGJ₂ not related to the PPAR activation, suggesting a more complex action of this prostanoid in tissues and in particular in the immune system (2, 3, 5).

In this regard, 15d-PGJ₂ is emerging as an interesting anti-inflammatory molecule functioning via PPAR-dependent and -independent mechanisms: it inhibits the production of inflammatory mediators such as TNF-, IL-1, iNOS, IL-12 by activated macrophages, microglial cells, and dendritic cells (6, 7, 8, 9, 10, 11); it may induce apoptosis of activated macrophages in the inflammatory site (12, 13); and more importantly, several in vivo studies support a role for 15d-PGJ₂ as a novel therapeutic agent, as observed in models of ischemia-reperfusion injury, inflammatory bowel disease (IBD), adjuvant-induced arthritis, and experimental autoimmune encephalomyelitis (EAE) (14, 15, 16, 17, 18).

Fas ligand (Fas-L) is a type II transmembrane protein able to trigger apoptotic cell death by binding to its receptor Fas (CD95) (19, 20), for which aberrant expression has been involved in diseases in which the peripheral lymphocyte homeostasis is compromised (21). Recently, a number of studies have revealed several pathophysiological roles of the Fas and Fas-L in vivo, particularly in the induction and regulation of certain organ-specific autoimmune diseases. In this regard, an improperly regulated Fas-Fas-L system could become a serious danger for the organism, leading to selective destruction of target cells within a tissue (22, 23). This process has been well documented in different animal models such as EAE or autoimmune diabetes in mice, in which the initial specific cell damage is mediated by tissue-infiltrating Fas-L⁺ T lymphocytes (22, 23). Moreover, additional functions for the Fas-Fas-L receptor-ligand pair have been described recently that expand the information available on its complex regulatory activity. In fact, triggering of Fas-L is required for CTLs to achieve an optimal proliferation (24, 25), and in addition, an activated Fas receptor can induce phenotypical and functional maturation of dendritic cells together with specific secretion of proinflammatory cytokines and a preferential local T cell polarization into a Th1 phenotype (26).

In the past few years, we have investigated the molecular mechanisms involved in normal physiological regulation and in pharmacological intervention applied to the modulation of cytokine expression in activated T lymphocytes. In particular, the role of specific activators of hormone nuclear receptors has been studied in these cells, and they were shown as useful and powerful modulators of proinflammatory cytokines and surface receptor ligands (27, 28, 29, 30).

In this report we describe the effect of the cyPG 15d-PGJ₂ on the activation of the fas-L gene in activated T lymphocytes and the regulatory actions of this prostanoid on the human fas-L promoter. We show that 15d-PGJ₂ suppresses fas-L mRNA expression and promoter activity in activated normal human T cells, in the 2B4.11 T cell hybridoma, and in Jurkat cells, and this inhibition correlates with a decreased activation-induced cell death (AICD) in these cells.

Promoter inhibition appears to be mediated by a mechanism independent of the PPAR nuclear receptor activity and not related to the activation of the heat shock factor (HSF)-1 and heat shock protein (HSP)70 stress response genes. Moreover, fas-L promoter analysis has identified a number of specific fas-L transactivators whose expression or function is differently modulated by 15d-PGJ₂. Among these, the expression of the early growth response (EGR)-3 factor and the transcriptional activity of Nur77 are strongly inhibited by 15d-PGJ₂.

Finally, the cyclopentenone reactive ring of 15d-PGJ₂, which is directly involved in this inhibitory mechanism as a molecular analog of 15d-PGJ₂ (CAY10410) with structural modifications intended to maintain PPAR ligand activity and to eliminate prostanoid metabolism via Michael addition to reactive nucleophiles, does not inhibit fas-L gene expression. In this regard, fas-L gene expression, promoter activation, and the specific transactivation driven by the fas-L enhancers AP-1, cMyc, NF-B, and Nur77 but not NF-AT are directly modulated by the reactive electrophile molecule cyclopentenone (2-cyclopenten-1-one) in activated T cells.

The data presented place fas-L as a novel molecular target to add to the variety of immunoregulatory activities mediated by 15d-PGJ₂. The physiological and pharmacological implications of these observations are discussed.

	Materials and Methods

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Cell lines and reagents

Jurkat cells and 2B4.11 murine T hybridoma cells were maintained as previously described (30). Human enriched T cells and T cell blasts were obtained from healthy donors as described (31). OKT3 anti-human CD3 mAb was purified from culture supernatant by protein A chromatography. PMA and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO). Cyclopentenone (2-cyclopenten-1-one) was purchased from Sigma-Aldrich. 15d-PGJ₂, ciglitazone, and cyclosporin A (CsA) were purchased from Biomol (Plymouth Meeting, PA). Rosiglitazone was purchased from ALEXIS (Lausen, Switzerland). CAY10410 (9,10-dihydro-15d-PGJ₂) was purchased from Cayman Chemicals (Ann Arbor, MI).

Assessment of cell viability and apoptosis

2B4.11 cells (5 x 10⁵/ml) were cultured in 24-well plates. Triplicate samples were stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin in the absence or in the presence of the indicated amount of 15d-PGJ₂ or 200 ng/ml CsA for 24 h in complete medium. Cells were then harvested and viability was assessed by addition of 5 µg/ml propidium iodide (PI) (Sigma-Aldrich) and immediate analysis by a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Dead cells were quantified as those taking up the dye. Apoptosis assay was performed by annexin V staining of the translocated phosphatidylserine from the inner side of the plasma membrane to the outer layer during the early stages of apoptosis. Triplicate samples of 2B4.11 cells (5 x 10⁵/ml) were cultured in 24-well plates and stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin in the absence or in the presence of 10 µM 15d-PGJ₂ or 200 ng/ml CsA for 5 h in complete medium. Cells were then stained using an Annexin V/FITC kit (Bender MedSystems, Vienna, Austria) following the manufacturer’s instructions and immediately analyzed by a FACScan flow cytometer (BD Biosciences).

Northern blot analysis

Total RNA was extracted from 2B4.11 hybridoma T cells or human T cells by TRIzol (Life Technologies, Grand Island, NY). Equal amounts of RNA (15 µg/lane) were fractioned on a 1.5% agarose-formaldehyde gel. The specific mRNA was detected by hybridization of S&S Nytran membranes (Schleicher & Schuell, Keene, NH) with a ³²P-labeled cDNA probe specific for the indicated gene. The RNA-containing membranes were prehybridized for 20 min and hybridized for 2 h at 65°C with the QuikHyb Hibridization Solution (Stratagene, La Jolla, CA). The membranes were then washed twice in 2x SSC containing 0.1% SDS and twice in 0.1x SSC containing 0.1% SDS at 60°C (20 min each time) and exposed to X-Omat AR films (Eastman Kodak, Rochester, NY) at -70°C with intensifying screens.

Plasmid constructions

The human Fas-L promoter luciferase reporter pFasL-486, the distal NFAT binding mutant (NFAT-Dist.), and the RE3/FLRE binding mutant were kindly provided by Dr. G. A. Koretzky (University of Pennsylvania, Philadelphia, PA) (32, 33). To prepare the human Fas-L promoter luciferase reporter pGL3-FasL-luc, the appropriate promoter fragment was subcloned into the HindIII site of the pGL3-basic luciferase vector (Promega, Madison, WI), as previously described for the pFasL-486Gal (27). The different deletions of the human Fas-L promoter -453 Fas-L(pGL2), -237 Fas-L(pGL2), and -195 Fas-L(pGL2) were kindly provided by Dr. C. V. Paya (Mayo Clinic, Rochester, MN) (31). The CMV--Gal expression vector pEQ176, the pGL3 Rous sarcoma virus (RSV)-luc, and the RSV-Gal expression vectors have been previously described (27). The expression vector for wild-type human PPAR (pSG5-PPAR) was kindly provided by Dr. B. Staels (Institut Pasteur de Lille, Lille, France). The expression vector for human PPAR (pcDNA31-L468A/E471A) dominant-negative mutant was kindly provided by Dr. V. K. K. Chatterjee (University of Cambridge, Cambridge, U.K.) (34). cDNAs for murine EGR-1, EGR-3, and Nur77 were kindly provided by Dr. J. Milbrandt (Washington University School of Medicine, St. Louis, MO). The cDNA for murine EGR-2 was kindly provided by Dr. P. Gilardi-Hebenstreit (Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale, Paris, France). cDNAs for murine and human Fas-L were kindly provided by Dr. Ruggero De Maria (Istituto Superiore di Sanitá, Rome, Italy). The human EGR-3 promoter luciferase reporter pRsa-luc was kindly provided by Dr. R. A. Kroczek (Robert Koch-Institute, Berlin, Germany) (35). Luciferase reporter vectors for the transcription factors AP-1 (pAP1-TA-Luc), cMyc (pMyc-TA-Luc), NF-B (pNF-B-TA-Luc), and NFAT (pNFAT-TA-Luc) and the control vector containing the minimal TATA-box from the HSV thymidine kinase promoter pTA-Luc were purchased by Clontech Laboratories (Palo Alto, CA). The expression vector for human Nur77/Nak1 was kindly provided by Dr. W. J. Kovacs (Vanderbilt University School of Medicine, Nashville, TN). The expression vector for rat Nurr1 was kindly provided by Dr. N. Ohkura (National Cancer Center, Tokyo, Japan). The luciferase reporter vectors for Nur77 (3xNurRE-Luc) and the control vector proopiomelanocortin (POMC-Luc basic) were kindly provided by Dr. J. Drouin (Institute de Recherches Cliniques de Montreal, Montréal, Québec, Canada). The human Hsp70B promoter -Gal reporter HSP70-Gal was purchased by StressGen Biotechnologies (Victoria, British Columbia, Canada). The expression vector for human constitutively activated HSF-1 S303A/S307A double-mutant (pcDNA3-HSF1-S303A/S307A) was kindly provided by Dr. R. I. Morimoto (Northwestern University, Evanston, IL). The expression vector for human hsp70 was kindly provided by Drs. Gerasimos N. Pagoulatos and C. E. Angelidis (University of Ioannina, Ioannina, Greece).

DNA transfections

Transfections of Jurkat cells were conducted by the DEAE-dextran method as described in Ref. 30 h, cells were treated with different combinations of stimuli, and after an additional 8 h, cells were harvested and protein extracts were prepared for the luciferase and -galactosidase assays as described in Ref. 30 . Protein concentration was quantified by the BCA method (Pierce, Rockford, IL). Luciferase activity was read using the luciferase assay system (Promega) following the manufacturer’s instructions. -Galactosidase activity was determined as described in Ref. 30 . 2B4.11 murine T hybridoma cells were transfected as described for Jurkat cells using 150 µg/ml DEAE-dextran. After 24 h, cells were treated with different combinations of stimuli for an additional 8 h and then processed as previously described.

EMSA

Nuclear proteins were prepared as described in Ref. 30 . Protein concentration of extracts was determined by the BCA method (Pierce). The nuclear proteins (10 µg) were incubated with radiolabeled DNA probes in a 20 µl reaction mixture containing 20 mM Tris (pH 7.5), 60 mM KCl, 2 mM EDTA, 0.5 mM DTT, 1–2 µg of poly(dI-dC), and 4% Ficoll. Where indicated, a molar excess of double-strand oligomer was added as a cold competitor, and the mixture was incubated at room temperature for 10 min before adding the DNA probe. Nucleoprotein complexes were resolved as described in Ref. 30 . Oligonucleotides were purchased by Invitrogen-Life Technologies (Groningen, The Netherlands). Complementary strands were annealed and end-labeled as described in Ref. 30 . Labeled DNA (3 x 10⁴ cpm) was used in a standard EMSA reaction. In supershift analysis, the specific Ab was added to the binding reaction, and the mixture was incubated for 30 min at room temperature before adding the labeled DNA probe. The Ab against HSF-1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An Ab against c-Jun (rabbit polyclonal; Santa Cruz Biotechnology) was used as a nonspecific Ab.

The following double-strand oligomers were used as specific labeled probes or cold competitors: Nur77 binding response element (NBRE), 5'-TCGAGTTTTAAAAGGTCATGCTCAATTTG-3'; heat shock element (HSE), 5'-agctCCTCGAATGTTCGCGAAGTTTCG-3'; and Octamer-(human histone H2b), 5'-agCTCTTCACCTTATTTGCATAAGCGAT-3'.

Western blot analysis

For Western blot analysis, cells were pelleted, washed once with cold PBS, resuspended in lysis buffer (1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, 10 µg/ml leupeptin, 20 µg/ml aprotinin in PBS), and subsequently incubated 30 min on ice. The lysate was centrifuged at 14,000 g for 15 min at 4°C, and the supernatant was collected as whole cell extract. Nuclear proteins were prepared as described in Ref. 30 . Protein concentrations of nuclear and cytoplasmic extracts were determined by the BCA method (Pierce).

For 2B4.11 and Jurkat cells, 30 µg of nuclear extract or whole cell extract were run on 12% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell) and blocked in 3% milk in TBST. Immunoreactive bands were visualized on the nitrocellulose membranes using HRP-coupled goat anti-rabbit or goat anti-mouse Ig and the ECL detection system (Amersham, Arlington Heights, IL) following the manufacturer’s instructions. Abs against Hsp70, EGR-3, EGR-1, RelA, Octamer-1, PPAR, c-Myc, and NF-ATc2 were purchased from Santa Cruz Biotechnology. The Ab against -actin was purchased from Sigma-Aldrich.

	Results

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15d-PGJ₂ inhibits fas-L gene expression and AICD in 2B4.11 murine T cell hybridoma

In this report, we investigated whether administration of 15d-PGJ₂ could affect gene induction and function of the fas-L gene in activated T cells. To this purpose, total RNA was isolated from 2B4.11 cells at 5 h after activation in the presence of 10 µM 15d-PGJ₂ and analyzed for fas-L mRNA expression by Northern blot assay. As shown in Fig. 1A, fas-L mRNA was induced by PMA plus ionomycin stimulation and strongly inhibited by the presence of the immunosuppressant CsA (used in the experiments of this report as a control for effective repression) and by 10 µM 15d-PGJ₂, indicating that fas-L is a molecular target of this prostanoid.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. 15d-PGJ2 represses fas-L mRNA expression and AICD in activated T cells. A, Northern blot analysis of total mRNA obtained from 2B4.11 hybridoma cells untreated (-) or stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin for 5 h, in the absence or in the presence of 200 ng/ml CsA or 10 µM 15d-PGJ2. The experiment shown is representative of various independent experiments all displaying similar results. B, A total of 5 x 105/ml of 2B4.11 hybridoma cells were stimulated in triplicate and activated with 20 ng/ml PMA and/or 0.5 µg/ml ionomycin in the absence or in the presence of 200 ng/ml CsA, or in the indicated concentration of 15d-PGJ2. At 24 h after stimulation, cells were harvested and viability was assessed by PI uptake and immediate analysis by a flow cytometer. Results are expressed as percent of apoptosis and represent the mean value (X ± SE) from at least four experiments. C, 2B4.11 hybridoma cells were stimulated as previously described for 5 h in the absence or in the presence of 10 µM 15d-PGJ2 or 200 ng/ml CsA. The cells stained with Annexin V-FITC single-positive (lower right) and PI/Annexin V-FITC double-positive (upper right) are early and late phase apoptotic cells, respectively. Unstimulated cells (upper left panel), 5 h PMA plus ionomycin (upper right panel), 5 h PMA plus ionomycin plus 10 µM 15d-PGJ2 (lower left panel), and 5 h PMA plus ionomycin plus 200 ng/ml CsA (lower right panel). Percent of single- and double-positive cells is displayed in each panel (lower right and upper right quadrants, respectively). The experiment is representative of four independent experiments all displaying similar results. D and E, Northern blot analysis of total mRNA obtained from fresh isolated-enriched human T cells (D) and from 3-day IL-2 cultured-enriched T cells (E), untreated (-) or activated by plate-bound anti-CD3 mAb (OKT3) for 6 h, in the absence or in the presence of 10 µM 15d-PGJ2.

The inhibition of fas-L mRNA expression was also confirmed in normal human T lymphocytes. As shown in Fig. 1, D and E, fas-L mRNA expression was significantly inhibited by 10 µM 15d-PGJ₂ in human fresh isolated-enriched T cells and in IL-2 culture-enriched T cells, activated by plate-bound anti-CD3 mAb (OKT3). Thus, activation-induced fas-L mRNA expression and AICD are inhibited by 15d-PGJ₂ in T cells, as previously shown for other modulators such as glucocorticoids, retinoids, and 1,25-(OH)₂D₃ (27, 37, 38).

fas-L promoter activity is inhibited by 15d-PGJ₂ in activated T cells

To determine whether one of the mechanisms involved in 15d-PGJ₂-mediated inhibition of fas-L gene activation could be direct interference with the transcriptional activity of its promoter, transient transfection assays were performed in 2B4.11 and Jurkat T cells. As shown in Fig. 2A, PMA plus ionomycin treatment of 2B4.11 cells induced activation of a human fas-L promoter fragment consisting of 486 bp immediately 5' of the translational start site (27), and the presence of 10 µM 15d-PGJ₂ significantly repressed the promoter activity. As a control for the specificity of action of 15d-PGJ₂ in the transfection system used in this study, a luciferase reporter driven by the RSV-long terminal repeat promoter was used in different experiments. As shown in Fig. 2B, the PMA plus ionomycin inducibility of this reporter was not repressed, but rather increased by 15d-PGJ₂, thus indicating that the repression observed on the fas-L promoter was not attributable to an aspecific effect on transcription. Similarly, 15d-PGJ₂ repressed activation of the fas-L promoter in Jurkat cells (Fig. 2, C and D), confirming the data also in a human T cell line. Thus, activation-induced fas-L promoter activity is inhibited by 15d-PGJ₂ in activated T cells.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. fas-L promoter activation is modulated by 15d-PGJ2. A and B, 2B4.11 cells were cotransfected with 20 µg of pGL3-FasL-luc or 10 µg of pGL3-RSV-luc, plus 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. At 24 h after transfection, cells were left untreated (-) or were stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) in the absence or in the presence of 10 µM 15d-PGJ2. After 8 h, cells were harvested and protein extracts were prepared for the luciferase and -galactosidase assays. Results are expressed as relative luciferase activity normalized to protein concentration as well as to -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least four experiments. C and D, Jurkat cells were transfected with 20 µg of pFasL-486Luc or 10 µg of pGL3-RSV-luc, plus 4 µg of pEQ176 CMV--Gal expression vector. Activation and samples harvesting were conducted as for 2B4.11 cells. Results are expressed as previously described and represent the mean value (X ± SE) from at least four experiments.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. 15d-PGJ2-mediated fas-L promoter repression and the role of PPAR. A and B, 2B4.11 cells were cotransfected with 20 µg of pGL3-FasL-luc reporter plus 4 µg of pSG5 empty expression vector or 4 µg of pSG-PPAR and 4 µg of pEQ176 CMV--Gal as previously described. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as relative luciferase activity normalized to protein concentration as well as to -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments. C–E, Jurkat cells were cotransfected with 20 µg of pFasL-486Luc reporter plus 4 µg of the indicated expression vector and 4 µg of pEQ176 CMV--Gal as previously described. Activation and samples harvesting were conducted as described. Results are expressed as relative luciferase activity normalized to protein concentration as well as to -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments. The percentage of specific repression relative to the control PMA plus ionomycin-triggered luciferase activity in each series in the absence of 15d-PGJ2 (considered as 100%) was 40% (A), 43% (B), 40% (C), 40% (D), and 52% (E). F, Western blot assay of total cellular proteins from Jurkat and 2B4.11 cells untreated (-) and stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) for 5 h, transiently transfected with the indicated expression vector (control for the anti-PPAR Ab specificity) or without transfection. The arrow indicates the specific PPAR band of the positive control. G and H, 2B4.11 and Jurkat cells were transfected with 20 µg of the indicated reporter vector and 4 µg of pEQ176 CMV--Gal as previously described. At 24 h after transfection, cells were left untreated (-) or were stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) in the absence or in the presence of 1 µM rosiglitazone or 10 µM ciglitazone. After 8 h, cells were harvested and protein extracts were prepared for luciferase and -galactosidase assays. Results are expressed as relative luciferase activity as previously described and represent the mean value (X ± SE) from at least three experiments.

Recent studies on the regulation of the fas-L promoter have identified several transactivators that cooperate in the transcription of this gene in activated T cells. Among these, NF-AT, EGR factors, and c-Myc play an absolutely necessary role in this process (31, 32, 33, 39, 40). Because 15d-PGJ₂ can regulate the activity of various signaling molecules and transcription factors (7, 8, 10, 41, 42, 43), we analyzed the effect of this prostanoid on the expression and/or nuclear translocation of transcription factors important for optimal fas-L promoter activity.

As shown in Fig. 4, treatment of PMA plus ionomycin-activated 2B4.11 cells with 15d-PGJ₂ inhibits the expression of EGR-3 as detected by Western blot assay of nuclear proteins. On the contrary, the level of EGR-1 was increased in the same context, while c-Myc, NF-AT, RelA, and the Octamer-1 transcription factors were not significantly altered. To confirm these data and investigate whether these effects were due to a specific regulation of the EGR genes, total RNA was isolated from 2B4.11 cells at 5 h after activation in the presence of 10 µM 15d-PGJ₂ and analyzed for egr-1, egr-2, and egr-3 mRNA expression by Northern blot assay. As shown in Fig. 5C, egr-3 mRNA was induced by PMA plus ionomycin stimulation and inhibited by the presence of 10 µM 15d-PGJ₂, indicating that the egr-3 gene is a molecular target of this prostanoid. On the contrary, 15d-PGJ₂ did not alter the expression of egr-2 (Fig. 5B) and surprisingly, it increased the expression of egr-1 (Fig. 5A). These data indicate that these related transcription factors are different when regulated by 15d-PGJ₂ in activated T cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. 15d-PGJ2 differently regulates EGR transcription factor expression. Western blot assay of nuclear proteins or total cellular proteins (for c-Myc) from 2B4.11 cells untreated (-) and stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) for 5 h in the absence or in the presence of 200 ng/ml CsA or 10 µM of 15d-PGJ2 as indicated. The different Western blots shown are representative of at least three independent experiments, all displaying similar results.

View larger version (53K): [in this window] [in a new window]   FIGURE 5. egr-3 mRNA and promoter activity are modulated by 15d-PGJ2. A–C, Northern blot analysis of total mRNA obtained from 2B4.11 hybridoma cells untreated (-) or stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin for 5 h in the absence or in the presence of 200 ng/ml CsA or 10 µM 15d-PGJ2. The Northern blots are representative of various independent experiments all displaying similar results. D, Western blot assay of nuclear proteins from Jurkat cells untreated (-) and stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) for 5 h in the absence or in the presence of 10 µM of 15d-PGJ2. The Western blots are representative of at least three independent experiments, all displaying similar results. E, Jurkat cells were transfected with 20 µg of pEGR3-Rsa-luc vector as described in Materials and Methods. At 24 h after transfection, cells were left untreated (-) or were stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) in the absence or in the presence of 10 µM of 15d-PGJ2. After 8 h, cells were harvested and protein extracts were prepared for the luciferase and -galactosidase assays. Results are expressed as relative luciferase activity and represent the mean value (X ± SE) of three experiments.

Thus, the induction of the egr-3 gene and its promoter activity is inhibited by 15d-PGJ₂ in activated T cells, suggesting that an altered expression and function of the EGR transcription factors might be involved in the repression of the of fas-L gene and promoter by this prostanoid.

Promoter analysis of 15d-PGJ₂-mediated fas-L inhibition

We investigated the possible presence of fas-L promoter region(s) involved in the repression mediated by 15d-PGJ₂. To this aim, we analyzed the activity of specific internal mutations and progressive deletions of the fas-L promoter by transient transfection assay. In these experiments, Jurkat cells were used as a convenient cell system for transfection assays as the transfection efficiency is higher in these cells in comparison to 2B4.11 cells. Transfection of fas-L promoter constructs bearing internal mutations that abrogate binding of critical transactivators, such as NF-AT (NFAT-Dist.) or EGR factors (RE3/FLRE), considerably decreased the inducible activation following stimulation with PMA plus ionomycin as previously reported (32, 33, 39). However, the specific percentage of repression of the residual promoter activity in the presence of 15d-PGJ₂ was not significantly altered in comparison to the wild-type fas-L promoter reporter (Fig. 6, A–C). Moreover, progressive deletion of these enhancer elements could delineate a minimal promoter fragment spanning nucleotides from -195 bp immediately 5' of the translational start site, which was still sensitive to 15d-PGJ₂ (Fig. 6, D–F). These data suggest that 15d-PGJ₂ can inhibit fas-L promoter activation by interfering with the enhancer activity of its first -195 bp immediately 5' of the translational start site.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. A promoter analysis of 15d-PGJ2-mediated fas-L inhibition. A–F, Jurkat cells were transfected with 20 µg of the indicated expression vector plus 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as relative luciferase activity normalized to protein concentration as well as to -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least four experiments. The percentage of specific repression relative to the control PMA plus ionomycin-triggered luciferase activity in each series in the absence of 15d-PGJ2 (considered as 100%) was 42% (A), 44% (B), and 60% (C).

View larger version (21K): [in this window] [in a new window]   FIGURE 7. The cyPG 15d-PGJ2 modulates the activity of specific fas-L promoter transactivators. A–E, Jurkat cells were cotransfected with 10 µg of the indicated reporter vector plus 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as percent activation relative to the control PMA plus ionomycin-triggered luciferase activity in each cotransfection series in the absence of 10 µM 15d-PGJ2 (considered as 100%) and represent the mean value (X ± SE) from at least four experiments. The histogram corresponding to the TATAmin-Luc control vector (E) is shadowed in the figure.

Because cyPGs (including their precursor arachidonic acid) have been shown to activate heat shock stress response genes in different experimental models (46, 47, 48), and because previous work has clearly shown that HSF-1 activation and HSP70 protein expression repress promoter activity and transcription of proinflammatory genes (49, 50, 51, 52), we investigated whether activation of the heat shock stress response by 15d-PGJ₂ could modulate the activity of the fas-L promoter in T cells. As shown in Fig. 8A, activation of 2B4.11 cells in the presence of 15d-PGJ₂ induces a specific DNA binding activity of HSF-1 as detected in mobility-shift assay, and this induction was functionally correlated to a production of the HSP70 protein as detected by Western blot assay (Fig. 8B). Similar observations were also obtained in Jurkat cells (data not shown). To determine whether 15d-PGJ₂-mediated inhibition of the fas-L promoter activity could be the result of a direct interfering action mediated by HSF-1 and/or HSP70 protein, transient transfection assays were performed in Jurkat cells with an expression vector encoding a constitutively active mutant of the human HSF-1. As shown in Fig. 8C, the expression vector encoding the constitutively active mutant of the human HSF-1 was functional, as it could activate a reporter vector driven by the human HSP70 promoter in PMA plus ionomycin stimulated Jurkat cells. However, overexpression of the active HSF-1 failed to modulate fas-L promoter activity but rather significantly increased its activity (Fig. 8D), suggesting that the transcriptional activity of HSF-1 was not involved in the repression. Similarly, overexpression of the human HSP70 protein in the same experimental setting did not modulate promoter activation, as shown in Fig. 8E. Thus, these data indicate that 15d-PGJ₂-mediated activation/expression of the heat shock response genes HSF-1 and HSP70 are not directly involved in the molecular events that repress the fas-L promoter in T cells.

View larger version (32K): [in this window] [in a new window]   FIGURE 8. The heat shock response genes HSF-1 and HSP70 are not directly involved in 15d-PGJ2-mediated fas-L inhibition. A, EMSA was performed using a 32P-labeled HSE oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (-) or PMA/ionomycin-treated 2B4.11 cells (4 h) in the absence or in the presence of 200 ng/ml CsA or 10 µM 15d-PGJ2. Where indicated, 100 ng of specific or nonspecific cold competitor (Octamer human histone H2b) or purified anti-HSF-1 was added to the reaction mixture to confirm specificity. The arrow represents the DNA binding of HSF-1-specific complexes. The same amount of a purified nonspecific Ab (anti-Octamer-1) did not supershift or inhibit HSE bound complexes (data not shown). B, Western blot assay of total cellular proteins from 2B4.11 cells untreated (-) and stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) for 5 h in the absence or in the presence of 200 ng/ml CsA or 10 µM 15d-PGJ2. Specific bands corresponding to Hsp70 and -actin (used in this study as a control for an equal amount of protein loading) are indicated by arrows. The Western blots shown are representative of at least three independent experiments, all displaying similar results. C–E, Jurkat cells were cotransfected with 20 µg of the indicated reporter vector plus 4 µg of RSV-luc expression vector (for the HSP70--Gal reporter) or 4 µg of pEQ176 CMV--Gal expression vector (for the FasL-486Luc reporter) as described in Materials and Methods. Where indicated, 5 µg of an expression vector encoding a constitutive active form of HSF-1 (pcDNA-HSF1, constitutively activated), or a human Hsp70 protein (hHSP70), or the pcDNA3 empty control vector was added to the cotransfection setting. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as relative -galactosidase or luciferase activities normalized to protein concentration as well as to the appropriate reporter activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments.

Nur77 (also called NAK1, TR3, or NGFI-B) is an orphan nuclear receptor belonging to the steroid/thyroid/retinoid nuclear receptor superfamily, initially identified as an immediate early serum-induced gene and subsequently as a gene induced by activation of T lymphocytes (53, 54, 55). It has been shown that during the TCR-mediated death of T cell hybridomas, nur77 is induced and its DNA binding to NBRE (or NurRE) correlates with the onset of apoptosis (53, 54, 55). Moreover, dominant-negative mutants of Nur77 or antisense inhibition of its expression, inhibit apoptosis of TCR-activated T cells, whereas constitutive expression of nur77 can lead to massive thymocyte apoptosis in transgenic mice (53, 54, 55, 56).

Because Nur77 is involved in the signal transduction of AICD and because its transcriptional activity directly correlates with its apoptotic functions (57) and with fas-L gene expression in several models (58, 59, 60), we investigated whether 15d-PGJ₂-mediated inhibition of AICD correlated with a specific interference of Nur77 expression and transcriptional activity in activated T cells. As shown in Fig. 9A, nur77 mRNA is strongly induced by PMA plus ionomycin in 2B4.11 cells and completely inhibited by CsA as previously described (54). However, treatment with 15d-PGJ₂ did not inhibit but even increased its expression as detected by Northern blot assay. We also analyzed the binding activity of Nur77 to a NBRE probe by mobility-shift assay, using nuclear extracts from activated 2B4.11 cells treated with 15d-PGJ₂. As shown in Fig. 9B, activation of 2B4.11 cells induced an NBRE-specific binding complex (completely inhibited by CsA, data not shown), and treatment with 15d-PGJ₂ slightly inhibited DNA binding. As a control for equal proteins loading, the same amount of nuclear proteins was run in the presence of Octamer factor(s)-specific probe (Fig. 9C). We then evaluated the transcriptional activity of Nur77 in activated T cells by using a multicopy reporter vector (3xNurRE-luc) specific for Nur77-mediated transactivation in T cells (61). As shown in Fig. 9, D and E, transfection of Jurkat cells with a 3xNurRE-luc reporter vector led to a strong activation after PMA plus ionomycin treatment as compared with the control vector (POMC-luc Basic) that was completely abolished by CsA. Surprisingly, treatment with 15d-PGJ₂ potently inhibited this reporter vector in the same experimental setting, despite the weak effect of this prostanoid on the NBRE DNA binding activity previously described. These data indicate that the transcription function of Nur77 is a molecular target of 15d-PGJ₂ and that gene expression, DNA binding, and transcriptional function are distinct features of nur77 differently modified by 15d-PGJ₂ in T cells. We also confirmed the role of Nur77 on the fas-L promoter activation in our experimental setting by cotransfection of a NAK1/Nur77 expression vector in Jurkat cells. As shown in Fig. 9F, overexpression of Nur77 could increase the activity of the fas-L promoter in a dose-dependent manner, an effect also obtained with the Nurr1/TINUR orphan nuclear receptor (Fig. 9G), a related nuclear receptor family member that bears >90% homology in its DNA binding domain with Nur77, but differ in the N- and C-terminal regions (55). Thus, 15d-PGJ₂ potently inhibits Nur77-mediated transactivation in T cells and this may contribute to the overall fas-L promoter repression observed.

View larger version (38K): [in this window] [in a new window]   FIGURE 9. 15d-PGJ2 is a powerful inhibitor of Nur77-driven transactivation. A, Northern blot analysis of total mRNA obtained from 2B4.11 hybridoma cells untreated (-) or stimulated with 20 ng/ml PMA and 0.5 µg/ml ionomycin for 5 h in the absence or in the presence of 200 ng/ml CsA or 10 µM 15d-PGJ2. The Northern blot shown is representative of various independent experiments all displaying similar results. B and C, EMSA was performed using a 32P-labeled NBRE or an Octamer-(human histone H2b) oligonucleotide as a probe in the presence of nuclear extracts (10 µg), from unstimulated (-) or PMA/ionomycin-treated 2B4.11 cells (4 h) in the absence or in the presence of 10 µM of 15d-PGJ2. Where indicated, 100 ng of specific or nonspecific cold competitor (Octamer human histone H2b for NBRE binding) were added to the reaction mixture to confirm DNA binding specificity. D–G, Jurkat cells were cotransfected with 10 µg (D and E) or 20 µg (F and G) of the indicated reporter vector plus 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. Expression vectors encoding Nur77, Nurr1, or pRcCMV empty control were added to the cotransfection setting as indicated in the figure. The total amount of expression vector cotransfected (F and G) was adjusted to 4 µg where needed by adding pRcCMV empty vector. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as relative luciferase activity normalized to protein concentration as well as to the -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments.

A number of reports have described various actions mediated by 15d-PGJ₂ that are independent of PPAR activation (1, 3). In this regard, cyPGs are characterized by the presence of a cyclopentenone ring structure containing an electrophilic carbon (Fig. 10A). This ring system can react covalently by means of the Michael addition reaction with a number of nucleophiles such as cysteine residues in cellular proteins, glutathione, or free sulfhydryls and is essential for many of the biological functions of cyPGs (10, 41, 62, 63, 64). Because fas-L promoter activity is inhibited by 15d-PGJ₂ through a mechanism independent of PPAR action, we analyzed the potential role of the cyclopentenone ring system of this prostanoid by using a molecular analog of 15d-PGJ₂, CAY10410, with structural modifications intended to maintain PPAR ligand activity and give it resistance to metabolism via Michael addition across the unsaturated enone, to glutathione and to other reactive nucleophiles (62, 65, 66). As shown in Fig. 10B, although the presence of 10 µM 15d-PGJ₂ inhibited activation-induced fas-L mRNA expression in 2B4.11 cells, treatment with the same molar concentration of CAY10410 did not inhibit but rather significantly increased fas-L mRNA expression. Moreover, activation of 2B4.11 cells in the presence of cyclopentenone (2-cyclopenten-1-one), a compound that mimics several of the biologic activities of cyPGs by virtue of its chemically reactive -unsaturated carbonyl group (63, 64), inhibited fas-L mRNA expression as well, indicating that adduct formation by Michael addition plays an important role for inhibition. Cyclopentenone-mediated inhibition was evident at concentrations from 200 (data not shown) to 500 µM (used in these experiments), a concentration range previously described to be effective in other cellular models (10). To extend this observation on the transcriptional activity of the fas-L promoter, transient transfection assays were performed in Jurkat T cells in the presence of these molecules, and as shown in Fig. 10C, activation of the fas-L promoter was significantly inhibited by Cyclopentenone, whereas CAY10410 greatly enhanced promoter activation in accordance with the data of fas-L mRNA induction showed in Fig. 10B.

View larger version (30K): [in this window] [in a new window]   FIGURE 10. The cyclopentenone reactive ring modulates fas-L promoter activity. A, Structures of 15d-PGJ2, CAY10410 (9,10-dihydro-15-deoxy-12,14-PG J2), and cyclopentenone (2-cyclopenten-1-one). The positions of chemically reactive electrophilic carbons are indicated by asterisks. B, Northern blot analysis of total mRNA obtained from 2B4.11 hybridoma cells untreated (-) or stimulated for 5 h with 20 ng/ml PMA and 0.5 µg/ml ionomycin for 5 h in the absence or in the presence of 10 µM 15d-PGJ2 or 10 µM CAY10410 or 500 mM cyclopentenone (Cyclopent.). The Northern blot is representative of three independent experiments all displaying similar results. C, Jurkat cells were cotransfected with 20 µg of FasL-486Luc reporter and 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. Cells were activated with 20 ng/ml PMA and 0.5 µg/ml ionomycin in the presence of 10 µM 15d-PGJ2 or 500 mM cyclopentenone (Cyclopent.) or 10 µM CAY10410 as described in Fig. 2. Results are expressed as relative luciferase activity normalized to protein concentration as well as to -galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 11. Cyclopentenone modulates the activity of fas-L promoter transactivators. A–F, Jurkat cells were transfected with 10 µg of the indicated reporter vector plus 4 µg of pEQ176 CMV--Gal expression vector as described in Materials and Methods. Activation and samples harvesting were conducted as described in Fig. 2. Results are expressed as percent activation relative to the control PMA plus ionomycin-triggered luciferase activity in each cotransfection series in the absence of 500 µM cyclopentenone (Cyclopt.) (considered as 100%) and represent the mean value (X ± SE) from at least four experiments. The histogram corresponding to the 3xNurRE-Luc reporter vector (F), is shadowed in the figure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In T lymphocytes, fas-L gene expression and programmed cell death can be strongly inhibited by various agonists of specific nuclear hormone receptors such as corticosteroids or retinoids (37, 38). Previous work from our group has identified fas-L together with other proinflammatory cytokine genes as important targets of the steroid hormone 1,25-(OH)₂D₃ (vitamin D₃) (27, 30), which has helped to explain the molecular basis of the immunosuppressive effects of this hormone (together with a number of related nonhypercalcemic analogs) and to propose new therapeutic tools for different chronic inflammatory autoimmune diseases.

In this report, we describe the inhibitory effect of the cyPG 15d-PGJ₂ on the expression of the fas-L gene in activated T cells and analyze the molecular mechanisms involved at the transcriptional level. We have shown that 2B4.11 hybridoma T cells and normal human T cells activated in the presence of 15d-PGJ₂ have an impaired expression of fas-L mRNA that correlated with a weaker AICD process. The repression mediated by 15d-PGJ₂ was remarkable, as compared with the inhibition obtained in the presence of the immunosuppressor CsA, both at the level of the fas-L mRNA expression and as quantified on the annexin V single-positive early apoptotic cells (Fig. 1). Thus, fas-L mRNA expression is inhibited by 15d-PGJ₂ in T cells similarly to that described for other immunomodulators such as glucocorticoids, retinoids, and 1,25-(OH)₂D₃ (27, 37, 38).

Repression of the fas-L gene was correlated to a significant inhibition of its promoter activity as detected in 2B4.11 and in Jurkat T cells (Fig. 2) and did not involve the nuclear receptor PPAR. In fact, overexpression of a wild-type receptor or of a dominant-negative mutant of PPAR did not modify the percent of specific 15d-PGJ₂-mediated fas-L promoter repression (Fig. 3, A–E). Moreover, PPAR was not detectable in our T cell lines as shown by Western blot assay (Fig. 3F), and the specific PPAR ligands rosiglitazone and ciglitazone were ineffective for repression (Fig. 3, G and H).

Interestingly, 15d-PGJ₂ strongly inhibited the expression of the fas-L transactivator EGR-3 as detected by Western blot assay, but did not inhibit the expression or nuclear translocation of other transcription factors important for optimal fas-L promoter activation such as NF-AT or c-Myc, and surprisingly, it increased the expression of the related family member EGR-1 (Fig. 4). 15d-PGJ₂-mediated inhibition of the egr-3 gene was also confirmed by Northern blot assay (Fig. 5C), whereas the mRNA levels of the related family member egr-2 were unaffected and the mRNA of the egr-1 gene significantly increased, accordingly to the data obtained by Western blot assay (Fig. 5, A and B). In this regard, the egr-3 promoter activity was significantly repressed in the presence of 15d-PGJ₂ in Jurkat cells (Fig. 5E), suggesting that transcription of the egr-3 gene is a target of 15d-PGJ₂ in T cells.

These data indicate that the EGR family of transcription factors are differently regulated by 15d-PGJ₂ and that this prostanoid does not aspecifically repress early gene activation in activated T cells. Moreover they suggest that a modified nonoptimal ratio of the EGR-1 and EGR-3 transcription factors might be involved in the repression of the of the fas-L gene.

Promoter mutational and deletion analysis of fas-L has indicated that the specific repression of normal and residual promoter activity by 15d-PGJ₂ was not significantly altered by mutations that abolished the activity of the critical enhancers NFAT-Distal (32) and FLRE (a DNA binding site for the EGR factors) (33, 39) (Fig. 6, A–C). Furthermore, progressive deletions of these enhancer elements could delineate a minimal promoter fragment spanning nucleotides from -195 bp immediately 5' of the translational start site, which was still sensitive to 15d-PGJ₂ (Fig. 6, D–F). These data indicate that the repressive mechanism(s) mediated by 15d-PGJ₂ on the fas-L promoter may involve additional regulatory factors other than EGR transactivators as previously suggested.

In this regard, the activity of the NF-B, cMyc, and to a lesser extent AP-1 transcription factors previously described to cooperate for optimal fas-L transcription (40, 44) was significantly repressed by 15d-PGJ₂. On the contrary, the activity of the NF-AT transcription factor was enhanced by 15d-PGJ₂ (Fig. 7D), suggesting that a number of activating and inhibitory events are simultaneously modified in T cells in the presence of this prostanoid that may modulate fas-L promoter activity at multiple levels.

Our Western blot assays (Fig. 4) and EMSA on nuclear extracts from 2B4.11 or Jurkat cells (data not shown) did not reveal any relevant increase of NF-AT nuclear translocation that might explain the positive effect on the activity of this transcription factor. A 15d-PGJ₂-mediated stimulation of the specific NF-AT transactivation capability may be suggested on the basis of our data. Additional experiments will be necessary to verify this hypothesis.

Recently, a PPAR-mediated inhibition of NF-B together with NF-AT and AP-1 DNA binding activity and repression of the transactivation driven by these transcription factors has been described in human T cells that correlated with inhibition of proliferation, IL-2 production, and promoter activation in the presence of synthetic and natural PPAR activators including 15d-PGJ₂ (67, 68₂ has been shown to induce IL-8 production via activation of mitogen-activated protein kinases together with the NF-B and AP-1 transcription factors (69). In this case the effect was not related to PPAR, as synthetic PPAR agonists did not mimic the 15d-PGJ₂-mediated IL-8 induction. The examples of these studies using purified peripheral blood T cells or different cell lines, together with many other reports done in various cellular models (Refs. 1, 2, 3 and references cited therein), highlight the complexity of action of this prostanoid in the immune system, showing in some cases opposite effects. In addition, mitogen-activated protein kinase activation can strongly inhibit PPAR activity, through direct phosphorylation of its amino-terminal A/B domain and a consequent negative intramolecular communication with the carboxyl-terminal ligand binding domain (Ref. 70 and references cited therein). These observations may then raise important questions on the specific role of PPAR as immunoregulator and should lead to reconsidering its action in the context of concomitant pathways triggered by various stimuli such as surface receptors (including Ag receptor), cytokines/growth factors, or other PGs.

One of the cellular responses activated by cyPGs is represented by the stress response, a complex array of signaling pathways activated by conditions that generate altered protein folding and processing, which induces the expression of various stress-related proteins including those of cytosolic hsp, such as HSP70, or its specific transactivator HSF-1. In this regard, the expression of HSP70 and HSF-1 by different stressors is able to regulate the transcriptional activation of several proinflammatory genes such as TNF- or IL-1 in various models (49, 50, 51, 52). In our experimental conditions, 15d-PGJ₂ could activate a strong stress response, as demonstrated by stimulation of HSF-1 DNA binding and transactivation (Fig. 8, A and B). However, overexpression of an active form of HSF-1 or of the HSP70 protein failed to modulate fas-L promoter activity (Fig. 8, C–E), suggesting that activation/induction of the stress response genes HSF-1 and HSP70 by 15d-PGJ₂ is not directly involved in the molecular events that repress the fas-L promoter in T cells.

To further analyze the molecular mechanisms involved in 15d-PGJ₂-mediated fas-L repression, we also tested the effect of this prostanoid on the expression, DNA binding, and transcriptional activity of Nur77, an early activated orphan nuclear receptor involved in TCR-induced death of T cells and able to regulate fas-L gene expression in different models (58, 59, 60). We found that 15d-PGJ₂ could strongly inhibit the PMA plus ionomycin-induced transactivation of a 3xNurRE reporter (a multicopy response element highly specific for Nur77 activity in T cells (61) (Fig. 9, D and E), and this inhibition was concomitant to an increased nur77 mRNA expression as detected by Northern blot assay (Fig. 9A). It has been shown that the cooperation of NF-AT together with the transcription factor MEF2D in a DNA binding-independent mechanism is a pivotal step for nur77 promoter activation in T cells (Ref. 55 and references cited therein). Our observation that the activity of NF-AT is enhanced by 15d-PGJ₂ (Fig. 7D) may suggest that such a mechanism might be involved to increase nur77 mRNA expression in this experimental system. 15d-PGJ₂ could only slightly reduce the activation-induced DNA binding ability of Nur factors to an NBRE-specific probe as detected by mobility-shift assay (Fig. 9B), an observation that does not explain the powerful repression of the 3xNurRE-driven activity. These data suggest that the transcriptional function of Nur77 represents a novel target of this prostanoid, although not related to its cellular expression or DNA binding ability to specific response elements.

The contribution of Nur77 to the fas-L promoter activity was verified in our experimental system, in which overexpression of NAK1/Nur77 or of the related family member Nurr1/TINUR used in this study as an additional control could increase promoter activity in a dose-dependent manner (Fig. 9, F and G), thus sustaining the indication that Nur77 may represent an additional molecular target for 15d-PGJ₂-mediated repression of fas-L transcription. Additional experiments will be necessary to understand how 15d-PGJ₂ may interfere with the activity of Nur77. A direct covalent modification of this orphan receptor by 15d-PGJ₂-mediated Michael addition that alters specific transcriptional functions might be hypothesized.

We have also verified the role of the cyclopentenone ring system of 15d-PGJ₂ by using one of its molecular analogs, CAY10410, a molecule with structural modifications that abolish its metabolism via Michael addition to reactive nucleophiles, thereby maintaining unaltered the PPAR ligand activity (Fig. 10A). T cell activation in the presence of CAY10410 could not inhibit but significantly increased fas-L mRNA expression in 2B4.11 cells, and similarly, it enhanced the promoter activity in Jurkat cells (Fig. 10, B and C). Moreover, the molecule cyclopentenone, which bears the chemically reactive -unsaturated carbonyl group (Fig. 10A) (63, 64), inhibited fas-L mRNA expression/promoter activation (Fig. 10, B and C) and modified the enhancer activity of the transcription factors NF-B, cMyc, AP-1, NF-AT, and NurRE similarly to that observed using 15d-PGJ₂ (Fig. 11), indicating that adduct formation by Michael addition plays an important role for inhibition.

The finding that CAY10410 may enhance the transcription of fas-L in these cell lines reveals that other pathway(s) triggered by 15d-PGJ₂ might be active simultaneously and be integrated to the final molecular events previously described. In this case, the cyclopentenone ring-mediated action might override the parallel activating pathway(s).

The exact mechanism of entry of 15d-PGJ₂ is still poor understood and it is possible that this prostanoid may enter in the cytoplasm by an active transporter system and be transported in the nucleus, thus affecting gene transcription. Alternatively, 15d-PGJ₂ might also act through a PG receptor as described for the CRTH2 G protein-coupled chemoattractant receptor-homologous molecule expressed in Th2 cells, basophils, and eosinophils (Ref. 1 and references cited therein). Moreover, all these mechanisms (including a possible PPAR activation) might not be mutually exclusive, rendering the mechanism of action of 15d-PGJ₂ complex to dissect.

In summary, this study has identified fas-L as a novel molecular target of 15d-PGJ₂ that extends the knowledge about the anti-inflammatory actions of this prostanoid. Repression of fas-L expression in activated T cells might act in combination with the various effects mediated by 15d-PGJ₂ at the inflammatory site, in particular at the onset of autoimmune diseases in which local inflammation and tissue injury mediated by Th1 cytokines and Fas-L-mediated killing can generate specific tissue and organ damage. The recent description of in vivo studies that reveal a role for 15d-PGJ₂ as a molecule that may ameliorate inflammation in diseases such as EAE or IBD (15, 18), together with the role mediated by Fas-L in these kinds of pathologies (23, 71), suggest that modulation of this gene by 15d-PGJ₂ in T cells may represent an additional tool to consider for treatment of specific autoimmune and inflammatory disorders.

	Acknowledgments

 
We thank Dr. Gary A. Koretzky and Dr. C. V. Paya for providing the human Fas-L promoter vectors, Dr. Bart Staels for providing the human PPAR expression vector, Dr. V. K. K. Chatterjee for the human PP