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The Journal of Immunology, 2007, 178: 4039-4050.
Copyright © 2007 by The American Association of Immunologists, Inc.

15-Deoxy-{Delta}12,14-Prostaglandin J2 Negatively Regulates rankl Gene Expression in Activated T Lymphocytes: Role of NF-{kappa}B and Early Growth Response Transcription Factors1

Cinzia Fionda2,*,{dagger}, Filomena Nappi§, Mario Piccoli*, Luigi Frati*,{ddagger}, Angela Santoni3,*,{dagger} and Marco Cippitelli3,4,*,{dagger}

* Department of Experimental Medicine and Pathology, Istituto Pasteur-Fondazione Cenci Bolognetti, University La Sapienza, Rome, Italy; {dagger} Regina Elena Cancer Institute, Centro Ricerca Sperimentale, Rome, Italy; {ddagger} Istituto Mediterraneo di Neuroscienze Neuromed, Pozzilli, Italy; and § National AIDS Centre, Istituto Superiore di Sanità, Rome, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptor activator of NF-{kappa}B ligand (RANKL) and its receptor RANK are cell surface proteins abundantly expressed in bone and lymphoid tissues, whose interaction triggers different signaling pathways leading to activation and differentiation of osteoclasts, pivotal actors of the normal bone remodeling cycle. Moreover, RANKL may act as an immunomodulator, representing an important dendritic cell survival factor produced by activated T cells. A large body of research has shown that not only does the RANKL/RANK system regulate the physiology of bone development but also plays an important pathological role in bone destruction mediated by inflammatory disorders or bone metastatic tumors. 15-Deoxy-{Delta}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. Although 15d-PGJ2 has been studied as a natural ligand of the peroxisome proliferator-activated receptor-{gamma} nuclear receptor, relevant peroxisome proliferator-activated receptor-{gamma}-independent actions mediated by this prostanoid have been described. In this study, we describe the effect of 15d-PGJ2 on the expression of the rankl gene in T lymphocytes. We show that 15d-PGJ2 inhibits rankl mRNA expression, protein, and rankl promoter activity by mechanisms mediated by its chemically reactive cyclopentenone moiety. Our data also indicate that 15d-PGJ2 represses rankl activation by interfering with the expression and/or activity of the transcription factors NF-{kappa}B, early growth response-2, and early growth response-3, whose altered balancing and transactivation may contribute for the repression of this gene. These results place rankl as a novel molecular target for the different immunoregulatory activities mediated by 15d-PGJ2. The physiological and pharmacological implications of these observations are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptor activator of NF-{kappa}B ligand (RANKL)5 (TNFSF11) is a type 2 transmembrane protein that belongs to the TNF family cytokines, produced by various tissues, and abundantly expressed in bone and lymphoid tissues (1). Although RANKL was originally identified as a dendritic cell survival factor produced by activated T lymphocytes (2, 3), additional studies have established a pivotal role of RANKL in the differentiation and activation of osteoclasts, multinucleate cells derived from the monocyte/macrophage hemopoietic lineage that are responsible for the resorption of mineralized bone during its development, homeostasis, and repair (1, 4, 5, 6).

In the last few years, a large body of evidence has shown that, in addition to the physiological homeostatic control of bone development and remodeling cycle, the RANKL/RANK system is also a major regulator of pathological bone destruction associated with different diseases such as rheumatoid arthritis, postmenopausal osteoporosis, periodontal diseases, and osteolytic cancer metastases (7, 8, 9, 10).

In this regard, a number of studies have clearly established the importance of T lymphocytes in the regulation of bone resorption; both activated CD4+ and CD8+ T cells secrete several cytokines that stimulate osteoclastogenesis such as TNF-{alpha}, IL-6, and IL-17. These cytokines, together the concomitant expression of RANKL by T cells, are important mediators of inflammatory bone diseases such as rheumatoid arthritis, periodontal diseases, and osteoporosis (11, 12, 13, 14, 15, 16). Moreover, T lymphocytes can also support osteoclastogenesis in in vitro models derived from human multiple myeloma (MM) bone disease; in addition, they can enhance the spontaneous osteoclastogenesis observed in PBMC cultures derived from patients with different tumor-induced osteolytic lesions because this process can be inhibited by selective depletion of T cells in the system (17, 18, 19). Therefore, the activity of T cells represents a relevant regulatory component of pathological bone remodeling.

15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), is a cyclopentenone-type PG (cyPG) of the J2 series deriving by dehydration of prostaglandin D2 (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 (20, 21).

This prostanoid is a natural ligand that activates the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor originally implicated in the regulation of adipogenesis (22) but also expressed by a variety of immune cells, including macrophages, T cells, B cells, and dendritic cells, in which it may regulate many aspects of their physiology (20, 21).

Moreover, an increasing number of reports have also described relevant regulatory mechanisms mediated by 15d-PGJ2 not related to the PPAR{gamma} activation, suggesting a more complex action of this prostanoid in tissues and in particular in the immune system (21, 23).

Several in vivo studies support a role for 15d-PGJ2 as an interesting immunomodulator, as observed in models of adjuvant-induced arthritis, ischemia-reperfusion injury, inflammatory bowel disease, and experimental autoimmune encephalomyelitis (24, 25, 26, 27, 28, 29).

In this context, 15d-PGJ2 represents an anti-inflammatory prostanoid working via PPAR{gamma}-dependent and -independent mechanisms; it inhibits the production of inflammatory mediators such as TNF-{alpha}, IL-1beta, inducible NO synthase, and IL-12 by macrophages, microglial cells, and dendritic cells (30, 31, 32, 33, 34), it induces apoptosis of macrophages in the inflammatory site (35), and it can inhibit the expression of IL-2 and Fas ligand in T lymphocytes (36, 37) or the functions of NK cells (38).

Recent studies have also reported that 15d-PGJ2 inhibits normal osteoclast formation and activity stimulated by osteoclastogenic cytokines such as RANKL; this effect correlates with a significant modulation of NF-{kappa}B (39), a transcription factor important for bone homeostasis and osteoclast differentiation but also involved in the onset and progression of inflammatory bone destruction, such as arthritis (40).

In this report, we describe the effect of the cyPG 15d-PGJ2 on the activation of the rankl gene expression in T lymphocytes and the regulatory actions of this prostanoid on the rankl promoter.

We show that, in activated 2B4.11 T cell hybridoma, 15d-PGJ2 suppresses RANKL expression with a significant inhibition of rankl mRNA expression, both in 2B4.11 hybridoma and in normal human T cells, and with the inhibition of rankl promoter activity in transiently transfected Jurkat cells.

Inhibition of RANKL by 15d-PGJ2 is independent of the PPAR{gamma} nuclear receptor activation and is mediated by the prostanoid chemically reactive cyclopentenone moiety.

Moreover, we identified several rankl promoter transactivators whose expression or function is differently modulated by 15d-PGJ2. In particular, the expression of NF-{kappa}B cRel, early growth response (EGR)-2, and EGR-3 is strongly inhibited by 15d-PGJ2.

The data presented in this manuscript indicate rankl gene as a molecular target of the immunoregulatory activities mediated by 15d-PGJ2. The physiological and pharmacological implications of these observations in the context of the novel field of the "osteoimmunology" are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and reagents

Jurkat T cells and 2B4.11 murine T hybridoma cells were maintained as described previously (41). Human-enriched T cells were obtained from healthy donors as described in Ref. 37 . OKT3 anti-human CD3 mAb was purified from culture supernatants by protein A chromatography. PMA and ionomycin were purchased from Sigma-Aldrich. Anti-murine CD3 {epsilon}-chain mAb (clone 145-2C11) was purchased by BD Biosciences-Pharmingen. Cyclopentenone (2-cyclopenten-1-one) was purchased from Sigma-Aldrich. 15d-PGJ2, cyclosporin A (CsA), Bay 11-7085, MG132, and SN50 were purchased from Biomol. 9,10-Dihydro-15-deoxy-{Delta}12,14-prostaglandin J2 (CAY10410) was purchased from Cayman Chemical.

Flow cytometric analysis

After stimulation, 1 x 106 2B4.11 cells were washed twice with PBS and incubated for 20 min at 4°C with 0.25 µg of a PE-conjugated rat anti-murine RANKL (PE anti-mouse RANKL, clone IK22/5) (Biolegend), following the manufacturer’s instructions. After an additional wash with PBS, cells were analyzed using a flow cytometer FACSCalibur (BD Biosciences).

RNA isolation and RT-PCR

Total RNA was extracted from 2B4.11 hybridoma T cells or human T cells by TRIzol (Invitrogen Life Technologies). One to 2 µg of total RNA was reverse transcribed (Promega), and aliquots were used in subsequent PCR. Primer sets are as follows: human rankl sense, 5'-agcacatcagagcagagaaagc-3', and antisense, 5'-cagtaaggaggggttggagacc-3'; murine rankl sense, 5'-ccgagactacggcggatcctaaca-3', and antisense, 5'-tcagtctatgtcctgaactttgaaagcccc-3'; murine c-rel sense, 5'-agagaaggggaatgcgttttagataca-3', and murine c-rel antisense, 5'-caggaaggaaaaacatgaaaacaca-3'; and beta-actin sense, 5'-gtggggcgccccaggcacca-3', and beta-actin antisense, 5'-ctccttaatgtcacgcacgatttc-3'. Semiquantitative PCR conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction.

Plasmid constructions

The murine rankl promoter luciferase reporters –2-kb murine (m) mRANKL/Luc and –110-bp mRANKL/Luc (in pGL3-basic luciferase vector; Promega) were provided by Dr. X. Fan (Emory University Medical School, Atlanta, GA). The human rankl promoter luciferase reporter –2-kb human RANKL/Luc (in pGL2-basic luciferase vector; Promega) was provided by Dr. S. V. Reddy (Medical University of South Carolina, Charleston, SC). The Rous sarcoma virus (RSV)-Gal and RSV-Luc expression vectors have been described previously (41). The expression vector for the dominant-negative mutant of the repressor IkB{alpha} (S32A and S36A) (in pRcCMV; Invitrogen Life Technologies) was provided by Dr. A. Israel (Institut Pasteur, Paris Cedex, France). The retroviral vector for the dominant-negative mutant of the repressor IkB{alpha} (2N{Delta}4) (in pMSCVneo; BD Clontech) was provided by Dr. J. Hiscott (Universit{iota} McGill University, Montreal, Quebec, Canada). To construct the 4x mRANKL NF-{kappa}B/Luc reporter, four copies of the NF-{kappa}B binding site spanning the position from –431 to –422 bp were subcloned into the pTAL-Luc luciferase vector containing the minimal herpes simplex virus TK-promoter (BD Clontech). Expression vectors for EGR-1 and EGR-3 were provided by Dr. J. Milbrandt (Washington University School of Medicine, St. Louis, MO). The expression vector for EGR-2 was provided by Dr. P. Gilardi-hebenstreit (Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale, Paris, France).

DNA transfections

Transfections of Jurkat cells were conducted by the DEAE-dextran method as described in Ref. 41 . To decrease variations in the experiments due to different transfection efficiency, cells were transfected in single batches, which were then separated into different drug treatment groups. A RSV-Gal expression vector was cotransfected each time to normalize DNA uptake. After 24 h, cells were treated with different combinations of stimuli, and after additional 16 h, cells were harvested and protein extracts were prepared for the luciferase and beta-galactosidase assays as described in Ref. 41 . Protein concentration was quantified by the BCA method (Pierce). Luciferase activity was read using the luciferase assay system (Promega) following the manufacturer’s instructions. beta-Galactosidase activity was determined as described in Ref. 41 .

Virus production and in vitro transduction

Phoenix retrovirus packaging cell lines were cultured in DMEM plus 10% FBS. Phoenix cells were transfected with viral DNA (5 µg of pMSCVneo-IkB{alpha} 2N{Delta}4 or pMSCVneo) at 50% confluence with LipofectAMINE Plus (Invitrogen Life Technologies). After transfection, the cells were placed in fresh medium. After an additional 24-h culture, virus-containing supernatant was harvested, filtered, and either stored at –20°C or used immediately for infection. Infection was performed on 0.5 x 106 2B4.11 cells in 3 ml of complete medium with Polybrene (8 µg/ml) (hexadimethrine bromide; Sigma-Aldrich) for 8–12 h. After infection, cells were allowed to expand for 48 h and were then selected for neomycin resistance. Amount of G418 used in selection was 500 µg/ml.

EMSA

Nuclear proteins were prepared as described in Ref. 41 . 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(deoxyinosinic-deoxycytidylic acid), 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. 41 . Oligonucleotides were purchased by Invitrogen Life Technologies. Complementary strands were annealed and end labeled as described in Ref. 41 . Approximately 3 x 104 cpm of labeled DNA 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 Abs against RelA, cRel, p50, Egr-1, Egr-2, and Egr-3 were purchased from Santa Cruz Biotechnology.

The following double-strand oligomers were used as specific labeled probes or cold competitors (sense strand): mRANKL NF-{kappa}B, 5'-gagttctagaatttccccaagtctt-3'; mRANKL –105/–81, 5'-aaggagggcagatgtgggagtgaaa-3'; NF-kB Ig enhancer, 5'-gatcacaagggactttccgct-3'; and octamer-(h-histone H2b), 5'-agctcttcaccttatttgcataagcgat-3'.

Western blot analysis

For Western blot analysis, 2B4.11 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, and Complete protease inhibitor mixture (Roche) in PBS), and subsequently incubated 30 min on ice. The lysate was centrifuged at 14,000 x g for 15 min at 4°C, and the supernatant was collected as whole-cell extract. Nuclear proteins were prepared as described above. Protein concentration of nuclear and whole-cell extracts was determined by the BCA method (Pierce). Thirty to 50 µg of nuclear extract or whole-cell extract was run on 12% denaturing SDS-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell) and blocked in 3% milk in TBST buffer. Immunoreactive bands were visualized on the nitrocellulose membranes, using HRP-coupled goat anti-rabbit or goat anti-mouse Igs and the ECL detection system (Amersham Biosciences), following the manufacturer’s instructions.

Abs against RANKL, cRel, RelA, and octamer-1 were purchased from Santa Cruz Biotechnology. The Ab against beta-actin was purchased by Sigma-Aldrich.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
15d-PGJ2 inhibits RANKL expression in 2B4.11 murine T cell hybridoma

Although an increasing number of studies is clearly demonstrating an important role of RANKL in the physiology and pathology of bone remodeling and immune system homeostasis (1), little is known about the regulation of rankl gene expression in resting and activated T lymphocytes. In this regard, the finding that, in the context of inflammation, activated T cells are able to induce and sustain pathologic osteoclast differentiation and activity has encouraged the research on therapeutic strategies aimed to target the unbalanced action of several bone-resorbing cytokines such as TNF-{alpha}, IL-17, and more importantly RANKL expressed by these cells (9).

In this study, we investigated whether the treatment with the prostanoid 15d-PGJ2 could affect the expression of RANKL in activated T cells.

2B4.11 hybridoma T cells were activated with PMA plus ionomycin in the presence or in the absence of 15d-PGJ2, and RANKL expression was measured by flow cytometric analysis. Activation of 2B4.11 T cells for 8 h induced RANKL, which was significantly decreased in the presence of 10 µM 15d-PGJ2 or in the presence of CsA (used in our experiments as a control for effective repression) (Fig. 1A). This result was also confirmed by Western blot analysis on whole-cell extracts obtained from 2B4.11 cells activated both by PMA plus ionomycin or immobilized anti-CD3 mAb, with or without 15d-PGJ2 (Fig. 1, B and C). Because activation of T cells leads to the activation of protein kinase C and mobilization of Ca2+, we have also examined in 2B4.11 cells the expression of RANKL in response to the single stimulus PMA, ionomycin, or their combination. As shown in Fig. 1D, only PMA alone but not ionomycin is able to weakly induce RANKL; however, in our experimental conditions, ionomycin synergized with PMA to induce RANKL, showing that concomitant activation of protein kinase C and Ca2+ mobilization is required to induce its optimal expression. 15d-PGJ2 is able to block the effect of these activators and inhibit RANKL expression in 2B4.11 cells activated by PMA alone and PMA plus ionomycin.


Figure 1
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  		FIGURE 1. 15d-PGJ2 represses RANKL expression in activated T cells. A, Flow cytometry analysis of cell surface RANKL expression. 2B4.11 cells were untreated (–) or stimulated with 10 ng/ml PMA and 0.5 µg/ml ionomycin for 8 h in the absence or in the presence of 10 µM 15d-PGJ2 or 200 ng/ml CsA. The experiment shown in the figure is representative of various independent experiments all displaying similar results. B–D, Western blot assay of total cellular proteins from 2B4.11 cells untreated (–) and stimulated with 10 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) (B), plate-bound anti-CD3 mAb (145-2C11) (C), or different stimuli combinations (D) for 16 h in the absence or in the presence of 10 µM 15d-PGJ2 as indicated in the figure. Arrows indicate the position of RANKL in the filters. E, Western blot analysis of total cellular proteins from 2B4.11 cells untreated (–) and stimulated with PMA + ionomycin (P/I) as described above for 16 h in the absence or in the presence of 10 µM 15d-PGJ2, 10 µM CAY10410, or 250 µM cyclopentenone (Cyclop.). The different Western blots shown in the figures are representative of various independent experiments, all displaying similar results.

 
This effect did not involve the action of the nuclear receptor PPAR{gamma} because CAY10410, a molecular analog of 15d-PGJ2 with structural modifications that abolish Michael addition to reactive nucleophiles, maintaining unaltered the PPAR{gamma} ligand activity, could not inhibit RANKL expression. On the contrary, the molecule Cyclopentenone, which bears the reactive {alpha},beta-unsaturated carbonyl group and mimics the action of different cyPG (33, 42), showed the same inhibitory effect on RANKL expression as for 15d-PGJ2 (Fig. 1E). Thus, adduct formation by Michael addition plays an important role for inhibition.

15d-PGJ2 inhibits rankl mRNA expression and promoter activation in T lymphocytes

We investigated whether the treatment with 15d-PGJ2 could affect mRNA expression of rankl in activated T cells.

To this purpose, total RNA was isolated from 2B4.11 cells at 6 h after activation in the presence of 10 µM 15d-PGJ2 and analyzed for rankl mRNA expression by RT-PCR assay. As shown in Fig. 2A, rankl mRNA was induced by PMA plus ionomycin stimulation and significantly inhibited by 10 µM 15d-PGJ2, indicating that rankl gene expression is a novel molecular target of this prostanoid. The inhibition of rankl mRNA induction was also confirmed in normal human T lymphocytes. As shown in Fig. 2B, rankl mRNA was significantly inhibited by 10 µM 15d-PGJ2 in highly enriched isolated human T cells activated by plate-bound anti-CD3 mAb.


Figure 2
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  		FIGURE 2. 15d-PGJ2 represses rankl mRNA expression and promoter activity in activated T cells. A and B, RT-PCR analysis of total mRNA obtained from 2B4.11 cells, untreated (–), or activated with PMA + ionomycin as described above (A), or plate-bound anti-CD3 mAb (OKT3) (B) for 6 h in the absence or in the presence of 10 µM 15d-PGJ2. RT-PCR shown in the figure are representative of various independent experiments all displaying similar results. C and D, Jurkat cells were cotransfected with 10 µg of the indicated RANKL/Luc reporter plus 4 µg of RSV-GAL expression vector as described in Materials and Methods. Twenty-four hours after transfection, cells were left untreated (–) or were stimulated with 10 ng/ml PMA and 0.5 µg/ml ionomycin (P/I) in the absence or in the presence of 10 µM 15d-PGJ2. After 16 h, cells were harvested, and protein extracts were prepared for the luciferase and beta-galactosidase assays. Results are expressed as relative luciferase activity normalized to protein concentration as well as to beta-galactosidase activity produced off the internal control plasmid and represent the mean value (X ± SE) from at least three experiments.

 
Thus, activation-induced rankl mRNA expression is inhibited by 15d-PGJ2 in T cells.

To determine whether one of the mechanisms involved in 15d-PGJ2-mediated inhibition could be a direct interference with the transcriptional activity of rankl gene promoter, transient transfection assays were performed in Jurkat cells. As shown in Fig. 2, C and D, the stimulation of Jurkat cells by PMA plus ionomycin induced the activation of a murine and human rankl promoter fragment consisting of 2 kb immediately 5' of the transcriptional start site, and the treatment with 10 µM 15d-PGJ2 could repress these promoters.

Thus, activation-induced rankl promoter activity is inhibited by 15d-PGJ2 in activated T cells.

NF-{kappa}B is a transcriptional activator of rankl gene in T cells: effect of 15d-PGJ2

Recent studies on the transcriptional regulation of rankl gene have identified several transactivators that cooperate to activate its transcription in osteoblasts (43, 44, 45, 46, 47). However, little is known about the regulation of rankl in activated T cells.

Among the different actions of cyclopentenone prostanoids on gene expression, the inhibitory effect of 15d-PGJ2 on inflammatory response gene induction is most clearly understood (48). In particular, the action of this prostanoid on the NF-{kappa}B transcription factor activity has been well investigated in different models, and there are abundant evidences implicating NF-{kappa}B as a major target for PPAR{gamma}-independent gene repression mediated by 15d-PGJ2 (33, 49, 50).

Because 15d-PGJ2 can inhibit rankl gene and promoter activity in T lymphocytes, we investigated whether NF-{kappa}B might represent a potential transcriptional enhancer for this gene repressed by this prostanoid. As shown in Fig. 3, A and B, three different inhibitors of NF-{kappa}B (Bay 11-7085, an IkB{alpha} phosphorylation inhibitor, MG132, a proteasome inhibitor, and SN50, an inhibitor of NF-{kappa}B nuclear translocation) inhibited the expression of RANKL in 2B4.11 cells activated by PMA or the combination of PMA plus ionomycin. Interestingly, MG132 also down-regulated the transcriptional activity of both the murine and human rankl promoter in transient transfection assays performed in Jurkat T cells (Fig. 3, C and D); furthermore, overexpression of a dominant-negative mutant of the repressor IkB{alpha} (S32A and S36A) blocked promoter activation (Fig. 4, A–D).


Figure 3
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  		FIGURE 3. Inhibitors of NF-{kappa}B represses RANKL expression and promoter activation in activated T cells. A and B, Western blot analysis of total cellular proteins from 2B4.11 cells untreated (–) and stimulated with PMA + ionomycin (P/I) or PMA alone as described above for 16 h in the presence of the indicated inhibitors of NF-{kappa}B. The cells were pretreated for 1 h before activation, and the concentration of the inhibitors was as follows: 5 µM Bay 11-7085, 3 µM MG132, and 10 µM SN50. C and D, Jurkat cells were cotransfected with 10 µg of the indicated RANKL/Luc reporter plus 4 µg of RSV-GAL expression vector as described above. Twenty-four hours after transfection, cells were left untreated (–) or were stimulated with PMA + ionomycin (P/I) in the absence or in the presence of 3 µM MG132. After 16 h, cells were harvested, and protein extracts were prepared for the luciferase and beta-galactosidase assays. Results are expressed as relative luciferase activity as described above and represent the mean value (X ± SE) from at least three experiments.

 

Figure 4
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  		FIGURE 4. IkB{alpha} dominant negative (D.N.) (S32A–S36A) inhibits rankl promoter activation in T cells. A–D, Jurkat cells were cotransfected with the indicated murine or human RANKL/Luc reporter as described above. Where indicated, 3 µg of an expression vector encoding: a D.N. mutant of IkB{alpha}, or the pRcCMV empty control vector, was added to the cotransfection setting. E, As a control for the specificity of the IkB{alpha} (D.N.), the activity of a luciferase reporter driven by the NF-{kappa}B-insensitive RSV-LTR was tested in the same cotransfection setting. Activation and samples harvesting were conducted as described above. Twenty-four hours after transfection, cells were left untreated (–) or were stimulated with PMA + ionomycin (P/I). After 16 h, cells were harvested, and protein extracts were prepared for the luciferase and beta-galactosidase assays. Results are expressed as relative luciferase activity as described above, and represent the mean value (X ± SE) from at least three experiments.

 
We then analyzed the sequence of the first 2 kb of the murine rankl promoter and identified a putative NF-{kappa}B binding site spanning the position from –431 to –422 bp (Fig. 5A). Gel shift assays, performed with a synthetic double-stranded oligonucleotide corresponding to this promoter element and nuclear extracts from activated 2B4.11 cells, demonstrated a retardation in the electrophoretic mobility due to a specific binding of NF-{kappa}B complexes, which contained the RelA, p50, and cRel proteins as demonstrated by supershift analysis (Fig. 5, B and C). Moreover, this NF-{kappa}B element could enhance basal transcription when multimerized 5' to a heterologous minimal promoter in a luciferase reporter vector transiently transfected in Jurkat cells (Fig. 5, D and E); in addition, NF-{kappa}B blockade by stable overexpression of a IkB{alpha} superrepressor down-regulated the expression of RANKL in 2B4.11 cells (Fig. 5, F and G).


Figure 5
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  		FIGURE 5. NF-{kappa}B binds to the murine rankl promoter. A, Schematic representation of the proximal (–1 kb) murine rankl promoter region with the putative NF-{kappa}B site identified. A comparison with a NF-{kappa}B binding "consensus" (underlined) is shown. B, EMSA was performed using the 32P-labeled mRANKL NF-{kappa}B oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h). Where indicated, 100 ng of specific NF-{kappa}B Ig or a nonspecific cold competitor (octamer h-histone H2b) was added to the reaction mixture to confirm specificity. C, Supershift analysis of NF-{kappa}B complexes bound to the mRANKL NF-{kappa}B oligonucleotide: where indicated, purified anti-RelA, anti-p50, or anti-cRel was added to the reaction mixture. The same amount of a nonspecific Ab (anti-octamer-1) did not supershift or inhibit NF-{kappa}B bound complexes (data not shown). D and E, Jurkat cells were cotransfected with 10 µg of RANKL/Luc multicopy reporter (or the control pTal/Luc vector) plus 4 µg of RSV-GAL expression vector. Activation and samples harvesting was conducted as described above. Results are expressed as relative luciferase activity as described above and represent the mean value (X ± SE) from at least three experiments. F, EMSA was performed as described above, in the presence of nuclear extracts (10 µg), from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h), stably transduced with the indicated retrovirus. G, Western blot analysis of total cellular proteins from the indicated stably transduced 2B4.11 cells untreated (–) and stimulated with PMA + ionomycin (P/I) for 16 h. The Western blot shown in the figure is representative of various independent experiments, all displaying similar results.

 
To analyze the effect of 15d-PGJ2 on NF-{kappa}B binding to this sequence, gel shift assays were performed with nuclear extracts prepared from 2B4.11 cells activated in the presence of increasing amounts of the prostanoid or cyclopentenone. Both compounds were able to significantly inhibit DNA binding of NF-{kappa}B complexes (Fig. 6A); as a control for equal proteins loading, the same amount of nuclear extract was run in the presence of an octamer factor(s)-specific probe.


Figure 6
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  		FIGURE 6. 15d-PGJ2 modulates NF-{kappa}B DNA binding and expression in T cells. A, EMSA was performed using the 32P-labeled mRANKL NF-{kappa}B oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h) in the absence or in the presence of increasing amounts of cyclopentenone (250–500 µM) or 15d-PGJ2 (7.5–15 µM). The same nuclear extracts were also used with an octamer factor(s)-specific probe as a control. The mobility of octamer-1 is shown in the figure. B and C, Western blot assay of nuclear proteins (n.ex.) or total cellular proteins (w.c.e.) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h) in the absence or in the presence of 10 µM 15d-PGJ2. The different Western blots shown in the figure are representative of at least three independent experiments, all displaying similar results. D, RT-PCR analysis of total mRNA obtained from 2B4.11 hybridoma cells, untreated (–), or activated with PMA + ionomycin (6 h) in the absence or in the presence of 10 µM 15d-PGJ2. The RT-PCR shown in the figure is representative of various independent experiments all displaying similar results.

 
This correlated with a significant inhibition of cRel (but not RelA) expression/nuclear translocation, as detected by Western blot assays on nuclear extracts or whole-cell extracts and by RT-PCR assay of c-rel mRNA (Fig. 6, B–D).

Therefore, NF-{kappa}B is a transcription factor involved in rankl gene and promoter activation significantly inhibited by 15d-PGJ2 in activated T cells.

EGR factors are transcriptional activators of rankl gene in T cells: effect of 15d-PGJ2

We have previously demonstrated that the expression of different EGR factors could be differently modulated by 15d-PGJ2 in activated T cells (37). The EGR family of transcription factors consists of four proteins with highly conserved zinc finger DNA-binding domains initially discovered as genes induced in response to different growth factors (51). EGR-1, EGR-2, and EGR-3 are induced by TCR engagement and regulate different genes in T lymphocytes. In this regard, EGR-1 may cooperate to the induction of IL-2 (52) and up-regulation of the IL-2Rbeta (53), and EGR-2 and EGR-3 have been characterized as important transactivators of TNF family genes such as fas-l (54, 55) or tnf{alpha} (56).

We then investigated whether EGR factors might regulate expression of rankl gene as described for fas-l. A computer analysis of the first 2 kb in the murine rankl promoter identified a putative EGR(s) binding site spanning the position from –92 to –85 bp immediately downstream to a tandem of SP1/SP3 binding elements recently described in the proximal promoter (46) (Fig. 7A). Interestingly, this putative binding site is almost identical to the human and murine EGR binding element of the fas-l gene (FLRE) (54). Gel shift assays performed with a synthetic double-stranded oligonucleotide spanning this promoter element (mRANKL –105/–81) and nuclear extracts from 2B4.11 cells stimulated with PMA plus ionomycin demonstrated a retardation in the electrophoretic mobility due to a specific binding of SP1 and activation-induced EGR complexes (Fig. 7B). Supershift analysis of the complexes indicated the presence of EGR-1, EGR-2, and EGR-3 bound to this promoter element (Fig. 7C), and more importantly, transient overexpression of EGR-2 or EGR-3 but not EGR-1 could significantly enhance the transcriptional activity of a –2 kb and (to a lesser extent) a minimal –110-bp rankl promoter deletion in transfected Jurkat cells (Fig. 8).


Figure 7
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  		FIGURE 7. EGR transcription factors bind to the murine rankl promoter. A, Schematic representation of the proximal (–1 kb) murine rankl promoter region, with the putative EGR(s) site identified. The EGR binding element of the human and murine Fas ligand promoter (FLRE) is also shown in the picture (box). B, EMSA was performed using the 32P-labeled mRANKL –105/–81 oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h). Where indicated, 100 ng of specific, FLRE, SP-1 Hiv, or a nonspecific cold competitor (octamer h-histone H2b) was added to the reaction mixture to confirm specificity. The mobility of SP-1 and of EGR factors is indicated by arrows in the figure. C, Analysis of EGR complexes bound to the mRANKL –105/–81 oligonucleotide: where indicated, purified anti-EGR-1, anti-EGR-2, or anti-EGR-3 was added to the reaction mixture. The same amount of a nonspecific Ab (anti-octamer-1) did not supershift or inhibit EGR bound complexes (data not shown). The asterisk indicates the position of a complex specifically competed by the cold oligonucleotide (as shown in B) and not supershifted by an anti-EGR-4 Ab (data not shown).

 

Figure 8
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  		FIGURE 8. EGR-2 and EGR-3 enhance the murine rankl promoter activity in T cells. A and B, Jurkat cells were cotransfected with 10 µg of the indicated RANKL/Luc reporter plus 4 µg of RSV-GAL expression vector. Where indicated, 4 µg of an expression vector encoding, EGR-1, EGR-2, EGR-3, or the pCB6 empty control vector, was added to the cotransfection setting. Activation and samples harvesting were conducted as described above. Twenty-four hours after transfection, cells were left untreated (–) or were stimulated with PMA + ionomycin (P/I). After 16 h, cells were harvested, and protein extracts were prepared for the luciferase and beta-galactosidase assays. Results are expressed as relative luciferase activity as described above and represent the mean value (X ± SE) from at least three experiments.

 
Therefore, EGR-2 and EGR-3 are transcription factors involved in rankl promoter regulation in activated T cells.

Interestingly, as shown in Fig. 9A, DNA binding of EGR-2 and EGR-3, but not EGR-1, were inhibited by 15d-PGJ2 (and by CsA used in this experiment as a control for effective repression), indicating that the action of the prostanoid may involve simultaneous modulation of different transactivators in the rankl promoter; as a control for equal proteins loading, the same amount of nuclear extract was run in the presence of an octamer factor(s)-specific probe (Fig. 9B). As we have demonstrated a role of NF-{kappa}B in rankl promoter activation, we wanted also to investigate its possible (indirect) action trough the regulation of other transactivators. We investigated whether NF-{kappa}B might influence the expression of EGR transcription factors in activated T cells. As shown in the EMSA of Fig. 9D, the NF-{kappa}B inhibitors Bay 11-7085 and MG132 could significantly repress NF-{kappa}B DNA binding activity to a canonical sequence of the Ig enhancer (used here as a positive control for inhibition) and, interestingly, repressed DNA binding of EGR-2 and EGR-3 but not EGR-1 on the mRANKL EGR element (Fig. 9E), indicating that NF-{kappa}B plays a role in the regulation of EGR-2 and EGR-3 expression/binding in T cells; as a control for equal proteins loading, the same amount of nuclear extract was run in the presence of an octamer factor(s)-specific probe (Fig. 9F).


Figure 9
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  		FIGURE 9. EGR transcription factors are differently modulated by 15d-PGJ2 and by inhibitors of NF-{kappa}B in T cells. A–C, EMSA were performed using a 32P-labeled mRANKL –105/–81 or an octamer oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h) in the absence or in the presence of 10 µM 15d-PGJ2 (A and B) or 200 ng/ml CsA (C). The mobility of SP-1, octamer-1 (Oct-1), and of EGR factors is indicated by arrows in the figures. D–F, EMSA were performed using a 32P-labeled NF-{kappa}B Ig, mRANKL –105/–81, or an octamer oligonucleotide as a probe in the presence of nuclear extracts (10 µg) from unstimulated (–) or PMA + ionomycin-treated 2B4.11 cells (6 h) in the absence or in the presence of 5 µM Bay 11-7085 or 3 µM MG132. The mobility of SP-1, Oct-1, and of EGR factors is indicated by arrows in the figures.

 
Therefore, the inhibitory action of 15d-PGJ2 on rankl gene transcription could involve a repressive mechanism on NF-{kappa}B activity that affect rankl promoter activation both directly and indirectly via concomitant inhibition of EGR-2/3 factors. In addition, 15d-PGJ2 could also inhibit EGR-2/3 expression/binding via different parallel mechanisms independent of NF-{kappa}B activity. Additional experiments will be necessary to clarify this hypothesis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Normal adult healthy bone undergoes tightly controlled dynamic and continuous remodeling. The net effect of bone remodeling is determined by the number and the activities of osteoclasts, bone-resorbing cells, osteoblasts, and bone-forming cells (57, 58). Nevertheless, excessive osteoclast activity is observed in different osteopenic disorders characterized by increased bone resorption and bone damage such as osteoporosis (8), Paget’s disease (59), and lytic bone metastases (10). In addition, generalized and local bone erosion has also been reported in chronic infections, such as hepatitis and HIV (60, 61), autoimmune and allergic diseases (62), or inflammatory arthritis (7), indicating that an activated immune system can affect bone homeostasis in different pathologies.

RANKL is a TNF family cytokine, which was identified as an essential factor for the induction of osteoclastogenesis (1, 4). This gene is also a major regulator of pathological bone destruction associated with different inflammatory diseases and osteolytic cancer metastases (7, 8, 9, 10).

RANKL is expressed by activated T lymphocytes (2, 3), and different studies have clearly established the importance of T cells in the regulation of bone remodeling. In fact, activated T cells secrete an array of cytokines that stimulate (e.g., TNF-{alpha}, IL-6, IL-17) or inhibit (e.g., IFN-{gamma}, IL-4, OPG) osteoclastogenesis. The net effect of T cells on osteoclast formation and action may consequently represent the prevailing balance of an anti- or a proosteoclastogenic pattern of secreted cytokines. In particular, in conditions such as inflammation, the effect of proosteoclastogenic cytokines prevails to activate pathologic bone erosion (11, 12, 13, 14, 15, 16).

In a different microenvironment, T lymphocytes can also support tumor-mediated osteoclastogenesis and bone destruction, as in the case of the MM, one of the most representative tumors able to generate devastating bone lesions. In this context, local T cells produce RANKL in response to IL-7 secreted by MM cells proliferating in the bone marrow and contribute to the generation of a critical imbalance of the RANKL/OPG system that leads to progressive bone erosion (17, 18, 63).

In this report, we describe the inhibitory effect of the cyPG 15d-PGJ2 on the expression of the rankl gene in activated T cells and analyze several regulatory 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 rankl mRNA, which correlated both with a lower protein expression and with a down-regulation of its promoter activity, as detected in Jurkat T cells transfected with the murine and the human promoter (Fig. 2).

This effect did not involve the activation of the nuclear receptor PPAR{gamma} and required the presence of the prostanoid reactive cyclopentenone ring. In fact, the molecule cyclopentenone, which bears the {alpha},beta-unsaturated carbonyl group and mimics the action of cyPG, showed similar inhibitory effects on RANKL expression as 15d-PGJ2 (Fig. 1E), indicating that adduct formation by Michael addition has a critical role for inhibition.

Little is known about the regulation of rankl in activated T cells, and abundant evidence implicate NF-{kappa}B as a major target for gene repression mediated by 15d-PGJ2 (33, 49, 50). Because 15d-PGJ2 can inhibit rankl gene expression and promoter activity in T lymphocytes, we investigated whether NF-{kappa}B might represent a potential transcriptional enhancer for this gene that could be repressed by this prostanoid. Indeed, we demonstrated that different inhibitors of NF-{kappa}B could strongly inhibit RANKL expression (Fig. 3, A and B), and furthermore, an IkB{alpha} superrepressor could suppress its promoter activation in transfected Jurkat cells (Fig. 4) and protein expression in stably transduced 2B4.11 cells (Fig. 5, F and G). Computer analysis of the first 2 kb of the murine promoter revealed the presence of a putative NF-{kappa}B binding site spanning the position from –431 to –422 bp (Fig. 5A). This NF-{kappa}B element could bind NF-{kappa}B in EMSA and could enhance basal transcription when multimerized 5' to a heterologous minimal promoter in a luciferase reporter vector (Fig. 5, B–E). 15d-PGJ2 (or cyclopentenone) was able to specifically inhibit DNA binding of NF-{kappa}B complexes to this enhancer element (Fig. 6A), and this correlated with a significant inhibition of cRel (but not RelA) expression and nuclear translocation, as indicated by Western blot assays on nuclear extracts or whole-cell extracts and by RT-PCR assay of c-rel mRNA (Fig. 6, B–D). These results indicate that NF-{kappa}B is a transcription factor involved in rankl gene and promoter activation and significantly inhibited by 15d-PGJ2 in T cells.

Interestingly, the computer analysis of the first 2 kb of the murine promoter revealed also the presence of a putative EGR(s) binding site spanning nucleotides from –92 to –85 bp, in a position immediately downstream to a tandem of SP1/SP3 binding elements, recently described in the proximal promoter (46) (Fig. 7A). This region is almost identical to the EGR binding site identified in the fas-l gene promoter (FLRE) (54, 55) (Fig. 7A), and gel shift assays detected a specific binding of SP1 and activation-induced EGR complexes associated with a DNA probe containing this promoter element (Fig. 7B). Supershift analysis of the induced complexes indicated the presence of EGR-1, EGR-2, and EGR-3 factors bound to this region (Fig. 7C), and more importantly, transient overexpression of EGR-2 or EGR-3, but not EGR-1, could significantly enhance the transcriptional activity of a –2 kb and (to a lesser extent) a –110-bp rankl promoter deletion in transfected Jurkat cells (Fig. 8).

In a previous study, we found that the expression of EGR factors could be differently modulated by 15d-PGJ2 in activated T cells (37); as shown in Fig. 9A, DNA binding of EGR-2 and EGR-3, but not EGR-1, was inhibited by 15d-PGJ2, indicating that the action of the prostanoid may involve simultaneous modulation of different transactivators of rankl promoter. Interestingly, the NF-{kappa}B inhibitors Bay 11-7085 and MG132 could significantly repress DNA binding of EGR-2 and EGR-3 but not EGR-1 on the mRANKL EGR element, suggesting that NF-{kappa}B is also involved in the regulation of EGR-2 and EGR-3 expression/binding in T cells (Fig. 9E). In this regard, in agreement with our data, inhibition of NF-{kappa}B has been shown recently to repress the expression of EGR factors in LPS-activated THP.1 monocytic cells (64). Therefore, the overall data presented here add novel information about the transcriptional regulation of rankl in T cells and show that the inhibitory action of 15d-PGJ2 on rankl gene transcription involves a repressive mechanism on NF-{kappa}B that could affect promoter activation both directly, by affecting the expression/activity of this transactivator, and indirectly via concomitant inhibition of EGR-2/3 factors. In addition, 15d-PGJ2 could also inhibit EGR-2/3 expression via different parallel mechanisms, independent of NF-{kappa}B activity. Additional experiments are required to clarify this hypothesis.

Bone-related diseases such as osteoporosis, rheumatoid arthritis, and cancer metastases affect millions of people worldwide, and the discovery of the RANKL/RANK genes as essential regulators of normal and pathologic bone remodeling has generated a great deal of attention toward novel molecules and therapeutic strategies aimed to target their pathological functions.

Interestingly, 15d-PGJ2 may directly inhibit osteoclast differentiation and activity stimulated by osteoclastogenic cytokines such as RANKL, and this effect correlates with a significant modulation of RANKL-induced NF-{kappa}B activation (39), a pivotal transcription factor for osteoclast differentiation, and involved in the onset and progression of inflammatory bone destruction (40).

By inhibiting RANKL expression in activated T cells, 15d-PGJ2 might significantly delay the progression of bone erosion in different examples of inflammatory pathologies (e.g., rheumatoid arthritis, periodontal diseases); moreover, this prostanoid might also ameliorate the frequent complications of cancers with bone metastasis that often results in severe disease and pain. In this regard, 15d-PGJ2 has already been shown to induce apoptosis in different types of tumor cells (20, 65). A good example of this action is MM, where the 15d-PGJ2-mediated suppression of NF-{kappa}B, an antiapoptotic regulator in these cells, is responsible for this mechanism (66). In this case, our results suggest that the presence of 15d-PGJ2 might simultaneously inhibit also the production of RANKL and other inflammatory cytokines (e.g., TNF) by conditioned activated T cells costimulated by a spectrum of local tumor-produced factors (such as IL-7) that directly or indirectly activate osteoclasts to resorb bone.

Thus, repression of RANKL in T lymphocytes might act in synergy with parallel effects mediated by 15d-PGJ2 at the inflammatory or the tumor site, in particular at the onset of bone destructive diseases, where local inflammation and tissue injury could generate specific conditions leading to bone damage and/or tumor progression.

Additional work will be needed to further characterize the effects of 15d-PGJ2 on RANKL expression/action and to support these data using nontransfected systems and in vivo models to improve our knowledge for a possible future application of this molecule (or novel prostanoids/Cyclopentenone derivatives) in the therapy, particularly in the context of pathological bone destruction associated with inflammatory diseases or osteolytic cancer metastases.


   Acknowledgments
 
We thank Drs. Xian Fan and Sakamuri V. Reddy for providing the murine and human rankl promoter luciferase vectors; Dr. A. Israel for providing the dominant-negative mutant of the repressor IkB{alpha} (S32A and S36A); Dr. J. Hiscott for providing the retroviral vector for the dominant-negative mutant of the repressor IkB{alpha} (2N{Delta}4); Dr. J. Milbrandt for murine EGR-1 and EGR-3 expression vectors; and Dr. P. Gilardi-hebenstreit for the murine EGR-2 expression vector.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was partially supported by grants from the Italian Association for Cancer Research, Ministero della Salute, 60% Ateneo, Progetti di Interesse Nazionale, Fondo per gli Investimenti della Ricerca di Base. Back

2 C.F. is recipient of a fellowship from the Italian Foundation for Cancer Research. Back

3 A.S. and M.C. contributed equally to this paper. Back

4 Address correspondence and reprint requests to Dr. Marco Cippitelli, Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, Rome, Italy. E-mail address: marco.cippitelli{at}uniroma1.it or cippitelli{at}ifo.it Back

5 Abbreviations used in this paper: RANKL, receptor activator of NF-{kappa}B ligand; CAY10410, 9,10-dihydro-15-deoxy-{Delta}12,14-prostaglandin J2; CsA, cyclosporin A; cyPG, cyclopentenone-type PG; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; EGR, early growth response; FLRE, fas-l regulatory element; MM, multiple myeloma; mRANKL, murine RANKL; PPAR, peroxisome proliferator-activated receptor; RSV, Rous sarcoma virus. Back

Received for publication September 22, 2006. Accepted for publication January 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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C. Fionda, F. Nappi, M. Piccoli, L. Frati, A. Santoni, and M. Cippitelli
Inhibition of Trail Gene Expression by Cyclopentenonic Prostaglandin 15-Deoxy-{Delta}12,14-Prostaglandin J2 in T Lymphocytes
Mol. Pharmacol., November 1, 2007; 72(5): 1246 - 1257.
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