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Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:710-716
Published online before print January 20, 2005, doi: 10.1161/01.ATV.0000156482.76228.d1
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:710.)
© 2005 American Heart Association, Inc.

Vascular Biology

15-Deoxy-{Delta}12,14-Prostaglandin J2 (15d-PGJ2) Signals Through Retinoic Acid Receptor–Related Orphan Receptor-{alpha} but Not Peroxisome Proliferator–Activated Receptor-{gamma} in Human Vascular Endothelial Cells

The Effect of 15d-PGJ2 on Tumor Necrosis Factor-{alpha}–Induced Gene Expression

Hideyuki Migita; John Morser

From the Department of Pharmacology (H.M.), Berlex Biosciences, Richmond, Calif; and Regenerative Medicine (H.M., J.M.),, Nihon Schering Research Center, Kobe, Japan.

Correspondence to Hideyuki Migita, Department of Pharmacology, Berlex Biosciences, Richmond, CA 94806. E-mail hideyuki_migita{at}berlex.com


*    Abstract
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 *Abstract
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Objective— 15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}), has been shown to inhibit proinflammatory gene expression, but the signaling mechanisms involved remain unclear. Because retinoic acid receptor–related orphan receptor-{alpha} (ROR{alpha}) has been reported to suppress tumor necrosis factor-{alpha} (TNF-{alpha})–induced expression of proinflammatory genes, we hypothesized that 15d-PGJ2 may induce ROR{alpha} expression resulting in inhibition of proinflammatory gene expression.

Methods and Results— We demonstrate that 15d-PGJ2 induced ROR{alpha}1 and ROR{alpha}4 expression and inhibited TNF-{alpha}–induced vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). In contrast, the synthetic PPAR{gamma} ligand pioglitazone weakly induced ROR{alpha}4 expression but did not affect ROR{alpha}1 expression or TNF-{alpha}–induced gene expression. Biphenol A diglycidyl ether, a PPAR{gamma} antagonist, did not block the effect of 15d-PGJ2 on ROR{alpha} expression. Adenovirus-mediated overexpression of ROR{alpha}1 inhibited TNF-{alpha}–induced VCAM-1 and ICAM-1 expression, and overexpression of a mutant form of ROR{alpha}1 (ROR{alpha}1{Delta}), which inhibited transcriptional activity of ROR{alpha}1 and ROR{alpha}4, attenuated its inhibition. Furthermore, we found that ROR{alpha}1{Delta} attenuated the inhibitory actions of 15d-PGJ2 on TNF-{alpha}–induced VCAM-1 and ICAM-1 expression.

Conclusions— These results suggest that 15d-PGJ2 inhibits TNF-{alpha}–induced expression of proinflammatory genes mediated in part via induction of ROR{alpha} in HUVECs. This mechanism provides a novel insight into PPAR{gamma}-independent actions of 15d-PGJ2.

15-Deoxy-{Delta}12,14-PG J2 (15d-PGJ2) inhibits proinflammatory gene expression, but its signaling mechanisms remain unclear. Because retinoic acid receptor–related orphan receptor-{alpha} (ROR{alpha}) suppresses proinflammatory gene expression, we hypothesized that 15d-PGJ2 induces ROR{alpha} expression, resulting in inhibition of proinflammatory gene expression. Our results provide a novel insight into anti-inflammatory actions of 15d-PGJ2.


Key Words: 15-deoxy-{Delta}12,14-prostaglandin J2 • retinoic acid receptor–related orphan receptor-{alpha} • peroxisome proliferator–activated receptor-{gamma} • endothelium • inflammation


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowDiscussion
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Vascular endothelial cells (ECs) are stimulated by proinflammatory cytokines such as tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) to express immunoglobulin superfamily proteins such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which interact with the {alpha}4ß1 integrin receptors and the ß2 integrin receptors of leukocytes, respectively.1,2 The expression of VCAM-1 and ICAM-1 is mediated by nuclear factor {kappa}B (NF-{kappa}B), which binds functional {kappa}B elements in their promoter/enhancer regions.3–5 Increased expression of adhesion molecules on ECs and adhesion of leukocytes to ECs are essential steps in vascular inflammation, such as atherosclerosis, as well as other chronic inflammatory diseases, such as arthritis and inflammatory bowel disease.6–9

Peroxisome proliferator–activated receptor-{gamma} (PPAR{gamma}) belongs to the superfamily of nuclear receptors and is a ligand-activated transcriptional factor, which regulates target genes by binding PPAR response elements in their promoter regions. A number of studies have demonstrated that PPAR{gamma} may play a role in regulating induced inflammatory responses.10–12 PPAR{gamma}-specific synthetic ligands, such as ciglitazone, troglitazone, pioglitazone, and rosiglitazone, have been shown to inhibit production of many inflammatory cytokines, such as TNF-{alpha}, IL-6, and IL-1ß, and expression of inducible NO synthase (iNOS) and matrix metalloprotease-9 in epithelial cells, monocytes, and macrophages,13–15 as well as to decrease TNF-{alpha}–induced EC apoptosis.16 The anti-inflammatory actions of PPAR{gamma} may be that it antagonizes the signaling pathways of NF-{kappa}B, activator protein-1 (AP-1), signal transducer and activator of transcription, or nuclear factor of activated T cells (NFAT).14,17,18 15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), the ultimate metabolite of prostaglandin (PG) D2, is best known as a natural ligand of PPAR{gamma}.19 It represses expression of iNOS and TNF-{alpha} in several cell types, whereas its modulation of inflammatory responses is, at least partially, dependent on PPAR{gamma}.14,20,21 However, there has been controversy over the existence of PPAR{gamma}-independent mechanisms for the anti-inflammatory actions of 15d-PGJ2. In the absence of PPAR{gamma} expression, 15d-PGJ2 also negatively regulates inflammatory responses.22,23 Recently, 15d-PGJ2 was shown to inhibit phorbol 12-myristate 13-acetate (PMA)– and lipopolysaccharide (LPS)-induced VCAM-1 expression in ECs,24 and 15d-PGJ2 and troglitazone were shown to inhibit TNF-{alpha}–induced expression of VCAM-1 and ICAM-1; but ciglitazone did not inhibit their expression.25 However, in another group study, no significant effects of 15d-PGJ2 and troglitazone on the TNF-{alpha}–induced VCAM-1 and ICAM-1 expression were reported.26 Therefore, the influence of 15d-PGJ2 on the inflammatory responses in ECs remains unclear and, in particular, the mechanisms of its actions are unknown.

Retinoic acid receptor–related orphan receptor-{alpha} (ROR{alpha}) also belongs to the nuclear receptor superfamily.27 The human ROR{alpha} gene encodes at least 4 distinct isoforms: ROR{alpha}1, ROR{alpha}2, ROR{alpha}3, and ROR{alpha}4, which only differ in their N-terminal domains. The 4 isoforms of ROR{alpha} display different binding preferences and ROR{alpha} response element (RORE)–dependent transcriptional activities. ROR{alpha}1 and ROR{alpha}4 are expressed in vascular cells, including ECs and smooth muscle cells (SMCs).28,29 Previous studies have demonstrated that ROR{alpha} suppresses TNF-{alpha}–induced expression of proinflammatory genes in vascular cells in part by inhibiting the NF-{kappa}B signaling pathway.29,30 Therefore, ROR{alpha} may play an important role in modulating inflammatory responses in vascular cells.

On the basis that 15d-PGJ2 exerts anti-inflammatory responses and ROR{alpha} suppresses TNF-{alpha}–induced expression of proinflammatory genes, we tested the hypothesis that 15d-PGJ2 induces ROR{alpha} expression and, if so, then 15d-PGJ2 will inhibit TNF-{alpha}–induced VCAM-1 and ICAM-1 expression via the induced ROR{alpha} in human ECs.


*    Methods
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 *Methods
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Cells and Reagents
Human umbilical vein ECs (HUVECs) were purchased from Clonetics. The cells were cultured in EBM-2 BulletKit (Clonetics). CV-1 cells (CCL-70; American Type Culture Collection) were cultured with Eagle’s minimum essential medium containing 10% FBS and nonessential amino acids. TNF-{alpha} was purchased from R & D Systems. 15d-PGJ2, pioglitazone, and biphenol A diglycidyl ether (BADGE) were obtained from Cayman Chemical. These compounds were dissolved in dimethyl sulfoxide (DMSO).

Real-Time RT-PCR Analysis
HUVECs were grown to confluent monolayers and treated with 15d-PGJ2, pioglitazone, or DMSO for 24 hours. For TNF-{alpha} stimulation, HUVECs were treated with 15d-PGJ2, pioglitazone, or DMSO for 22 hours, followed by stimulation with 0.2 ng/mL of TNF-{alpha} for 2 hours. For analysis of PPAR{gamma} dependence, HUVECs were treated for 24 hours with 15d-PGJ2, pioglitazone, or DMSO in the presence or absence of 3 µmol/L of BADGE. In these experiments, the final concentrations of DMSO in the culture medium were 0.1%. Then, total RNA was isolated from HUVECs with the RNeasy Mini Kit (Qiagen). RT-PCR analysis was performed essentially as described,29 using 15 ng total RNA and the GeneAmpEZ rTth RNA PCR Kit (Applied Biosystems). The specific primer sets for the target genes are as follows: for ROR{alpha}1, 5'-ACCCCGCTGAACCAGGAATC-3' and 5'-GAAGTTCCGTCAGCCCGTTG-3'; for ROR{alpha}4, 5'-CTCCGCACCGCGCTTAAT-3' and 5'-GAAGTTCCGTCAGCCCGTTG-3'. Quantitative real-time RT-PCR was performed with 30 ng of total RNA and the QuantiTect SYBR Green RT-PCR Kit (Qiagen).29,31 The specific primer sets for the target genes are as follows: for ROR{alpha}1, 5'-CGGTGCGCAGAC AGAGCTATT-3' and 5'-TTGTCTCCACAGATCTTGCATGG-3'; for ROR{alpha}4, CTCCGCACCGCGCTTAAAT-3' and 5'-TTGTCTCCACAGATCTTGCATGG-3'; for VCAM-1, 5'-TGGGCTGTGAATCCCCATCT-3' and 5'-GGGTCAGCGCGTGGAATTGGTC-3'; for ICAM-1, 5'-CGTGGGGAGAAGGAGCTGAA-3' and 5'-CAGTGCGGCACGAGAAATTG-3. RT-PCR was performed by incubating the reaction mixture for 30 minutes at 60°C and 15 minutes at 95°C, followed by 40 cycles of 15 seconds at 94°C, 30 seconds at 58°C, and 30 seconds at 72°C. GAPDH mRNA levels were measured with TaqMan EZ RT-PCR Kit (Applied Biosystems) and predeveloped TaqMan Assay Reagents Control Kit (Applied Biosystems) according to manufacturer instructions. The mRNA expression levels were normalized by GAPDH expression and presented as the relative expression level compared with the expression levels obtained from the control.

Transcription Assay
The pFA-PPAR{gamma} expression constructs were prepared using the ligand binding domain (LBD) of human PPAR{gamma} cDNA adjacent to the yeast GAL4 transcription factor DNA binding domain in the mammalian expression vector pFA-CMV (Stratagene). As a measure of cell-based GAL4-chimeric reporter activity assays, CV-1 cells were cotransfected using FuGENE6 (Roche) with 0.1 µg of pFA-PPAR{gamma} and 0.3 µg of pFR-luc (Stratagene). Transfected cells were cultured for 24 hours with Eagle’s minimum essential medium containing 2% FBS and nonessential amino acids. After treatment with 15d-PGJ2, pioglitazone, or DMSO for 24 hours, luciferase activity was measured (Dual Luciferase Assay Kit; Promega). The transcription activity of ROR{alpha} was performed essentially as described,29 with 1 µg of pTK-RORE-Luc plasmid, which contains a thymidine kinase promoter controlling the expression of luciferase with 4 tandem copies of RORE (TCGCAAAATGGGTCACGG), mixed with pcDNA encoding ROR{alpha}1 (0.1 µg), ROR{alpha}4 (0.3 µg), or ROR{alpha}1{Delta}. (0.2 µg). The total DNA amount of each group was adjusted to a total of 1.5 µg by addition of pcDNA. In these experiments, 0.02 µg of pRL-TK (Renilla luciferase reporter; Promega) was further included in the transfection mixture, and the promoter-dependent transcriptional activity was normalized to Renilla luciferase activity.

Western Blot Analysis
Cell extracts were prepared from HUVECs using CellLyticNu-CLEAR Extraction Kit (Sigma). The extracts (2.5 µg) were analyzed by Western blot analysis using specific antibodies against VCAM-1 (sc-13160) and ICAM-1 (sc-8439; Santa Cruz Biotechnology) and detected by chemiluminescence.

Adenovirus Infections
The recombinant adenoviruses expressing ROR{alpha}1 (Ad-ROR{alpha}1) and a mutant of ROR{alpha}1 (Ad-ROR{alpha}1{Delta}), which had the LBD deleted, were described previously.29 For adenovirus-mediated gene transfer, confluent HUVECs were exposed to Ad-ROR{alpha}1 (multiplicity of infection [moi] 10), Ad-ROR{alpha}1{Delta} (moi 20), or adenoviruses expressing ß-galactosidase (Ad-lacZ; Clontech; moi 10, 20, or 30). The moi of each group was adjusted to a total of 30 by addition of Ad-lacZ. After 6 hours, HUVECs were treated with 15d-PGJ2 or DMSO for 22 hours, followed by stimulation with 0.2 ng/mL of TNF-{alpha} for 2 hours. Then, total RNA was prepared and RNA analysis was performed.

Statistical Analysis
Quantitative data were expressed as means±SD from 4 to 6 experiments. Significance was determined either by Student t test for comparing 2 groups or by ANOVA, followed by Dunnet test for multiple comparisons. A value of P<0.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
15d-PGJ2 Induces ROR{alpha}1 and ROR{alpha}4 Expression
Two isoforms of ROR{alpha}, ROR{alpha}1 and ROR{alpha}4, are expressed in ECs.28,29 To understand the biological relevance of 15d-PGJ2 and ROR{alpha} in ECs, we first investigated whether 15d-PGJ2 affects ROR{alpha}1 and ROR{alpha}4 mRNA expression in HUVECs by RT-PCR analysis. 15d-PGJ2 induced ROR{alpha}1 expression and ROR{alpha}4 expression (Figure 1A). Quantitative real-time RT-PCR analysis demonstrated that 15d-PGJ2 induced ROR{alpha}1 and ROR{alpha}4 expression in a dose-dependent manner (Figure 1B and 1C). The maximal increases in ROR{alpha}1 expression (1.9-fold of the control) and ROR{alpha}4 expression (2.6-fold of the control) were observed with 3 µmol/L of 15d-PGJ2. In contrast, pioglitazone, a synthetic PPAR{gamma} agonist, did not significantly affect ROR{alpha}1 expression but induced ROR{alpha}4 expression (1.7-fold of the control). Transactivation assays showed that 15d-PGJ2 and pioglitazone were potent agonists of PPAR{gamma} (Figure 2), consistent with previous reports.21,32 Interestingly, pioglitazone and 15d-PGJ2 showed a similar dose dependency for PPAR{gamma} activation, although 10 µmol/L of pioglitazone had much less effect on ROR{alpha} expression than 3 µmol/L of 15d-PGJ2 (Figures 1 and 2 Down).



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  		Figure 1. 15d-PGJ2 induces ROR{alpha}1 and ROR{alpha}4 expression. A, HUVECs were treated with DMSO, pioglitazone (10 µmol/L), or 15d-PGJ2 (3 µmol/L) for 24 hours. Total RNA was prepared and analyzed by RT-PCR as described. B and C, HUVECs were treated with DMSO, pioglitazone (10 µmol/L), or 15d-PGJ2 (0.3, 1, or 3 µmol/L) for 24 hours. Total RNA was prepared and analyzed by quantitative real-time RT-PCR as described. The normalized expression of ROR{alpha}1 or ROR{alpha}4 to GAPDH is presented as the relative expression compared with the basal expression in DMSO-treated cells. cont indicates DMSO-treated control; PIO, pioglitazone. *P<0.05; **P<0.01 vs control.



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  		Figure 2. 15d-PGJ2 and pioglitazone are PPAR{gamma} agonists. CV-1 cells were transiently cotransfected with pFA-PPAR{gamma}, pFR-luc (Firefly), and pRL-TK (Renilla), cultured for 24 hours, and then incubated with DMSO, pioglitazone, or 15d-PGJ2 for 24 hours. Results are presented as fold induction of the basal activity of DMSO-treated cells. cont indicates DMSO-treated control; PIO, pioglitazone. *P<0.05; **P<0.01; ***P<0.001 vs control.

15d-PGJ2-Induced ROR{alpha} Expression Is Mediated by PPAR{gamma}-Independent Pathway
Because relevant PPAR{gamma}-independent actions mediated by 15d-PGJ2 have been described22,23,33,34 and 15d-PGJ2, in contrast to pioglitazone, clearly induced ROR{alpha} expression (Figure 1), we next examined whether 15d-PGJ2–induced ROR{alpha} expression is independent of PPAR{gamma} using BADGE, a PPAR{gamma} antagonist.35 BADGE completely attenuated the pioglitazone-induced ROR{alpha}4 expression in HUVECs (Figure 3B). However, BADGE affected neither the 15d-PGJ2–induced ROR{alpha}1 expression (Figure 3A) nor the 15d-PGJ2–induced ROR{alpha}4 expression (Figure 3B). These data show that ROR{alpha} expression was regulated by 2 independent signaling pathways: PPAR{gamma}-dependent and PPAR{gamma}-independent pathways and, in particular, the 15d-PGJ2–induced ROR{alpha} expression was mediated in part by the PPAR{gamma}-independent pathway.



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  		Figure 3. BADGE suppresses the pioglitazone-induced ROR{alpha}4 expression but does not alter the 15d-PGJ2–induced expression of ROR{alpha}1 (A) and ROR{alpha}4 (B). HUVECs were incubated with DMSO, pioglitazone (10 µmol/L), or 15d-PGJ2 (3 µmol/L) in the presence or absence of BADGE (3 µmol/L) for 24 hours. Total RNA was prepared and analyzed by quantitative real-time RT-PCR as described. The normalized expression of ROR{alpha}1 or ROR{alpha}4 to GAPDH is presented as the relative expression compared with the basal expression in DMSO-treated cells. PIO indicates pioglitazone. **P<0.01.

15d-PGJ2 Inhibits TNF-{alpha}–Induced VCAM-1 and ICAM-1 Expression
We next investigated the effect of 15d-PGJ2 on TNF-{alpha}–induced expression of proinflammatory genes. As shown in Figure 4A and 4B, stimulation by TNF-{alpha} highly induced VCAM-1 expression (930-fold of the basal expression) and ICAM-1 expression (380-fold of the basal expression) in HUVECs. We found that pretreatment of the cells with 15d-PGJ2 resulted in 51% inhibition of the TNF-{alpha}–induced VCAM-1 expression and 49% inhibition of the TNF-{alpha}–induced ICAM-1 expression. Interestingly, in contrast, pioglitazone did not significantly affect TNF-{alpha}–induced expression of the genes. The levels of VCAM-1 and ICAM-1 protein expression were confirmed by Western blotting analysis (Figure 4C).



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  		Figure 4. 15d-PGJ2 inhibits TNF-{alpha}–induced expression of VCAM-1 (A and C) and ICAM-1 (B and C). HUVECs were treated with DMSO, pioglitazone (10 µmol/L), or 15d-PGJ2 (3 µmol/L) for 22 hours. Then the cells were stimulated by TNF-{alpha} (0.2 ng/mL) for 2 hours. Quantitative real-time RT-PCR was performed (A and B), and Western blot analysis was performed (C) as described. The normalized expression of VCAM-1 or ICAM-1 to GAPDH is presented as the relative expression compared with the basal expression in nonstimulated cells. PIO indicates pioglitazone. *P<0.05 vs DMSO.

Dominant-Negative ROR{alpha}1 Attenuates the Inhibitory Effects of 15d-PGJ2 on TNF-{alpha}–Induced VCAM-1 and ICAM-1 Expression
We reported previously that ROR{alpha} negatively regulates TNF-{alpha}–induced expression of proinflammatory genes in ECs.29 Based on that earlier report and that 15d-PGJ2 induced ROR{alpha} expression (Figure 1) and inhibited TNF-{alpha}–induced VCAM-1 and ICAM-1 expression (Figure 4), we hypothesized that 15d-PGJ2–induced ROR{alpha} expression causes inhibition of TNF-{alpha}–induced VCAM-1 and ICAM-1 expression in HUVECs. The hypothesis was examined using the mutant form of ROR{alpha}1 (ROR{alpha}1{Delta}), which has the LBD deleted, lacked transcription activity, and attenuated transcription activities of ROR{alpha}1 and ROR{alpha}4 (Figure 5). To further understand the dominant-negative effect of ROR{alpha}1{Delta}, we examined whether ROR{alpha}1{Delta} attenuates the biological functions of ROR{alpha}1 in HUVECs. The experiment was performed using the ROR{alpha}1 isoform, because the transcription activity of ROR{alpha}1 was much stronger than that of ROR{alpha}4 and the potency as an anti-inflammatory was likely to depend on the transcription activities,29 suggesting ROR{alpha}1 is the functionally dominant isoform in ECs. As shown in Figure 6A and 6B, adenovirus-mediated gene transfer of ROR{alpha}1 to HUVECs led to decreases in TNF-{alpha}–induced VCAM-1 and ICAM-1 expression. In contrast, the infection of adenoviruses expressing ROR{alpha}1{Delta} to HUVECs did not affect the TNF-{alpha}–induced expression of either gene. The overexpression of ROR{alpha}1 and ROR{alpha}1{Delta} in HUVECs was confirmed by RT-PCR analysis and Western blotting analysis (data not shown).29 We found that ROR{alpha}1{Delta} attenuated the negative regulation of TNF-{alpha}–induced VCAM-1 and ICAM-1 expression by ROR{alpha}1. These data suggest that ROR{alpha}1{Delta} has a dominant-negative effect on the biological functions of ROR{alpha}1 as well as ROR{alpha}4 in HUVECs. Furthermore, as expected, ROR{alpha}1{Delta} attenuated the effects of 15d-PGJ2 on TNF-{alpha}–induced VCAM-1 and ICAM-1 expression (Figure 6A and 6B). As shown in Figure 6C, the effects of 15d-PGJ2 on VCAM-1 and ICAM-1 protein expression were also blocked by ROR{alpha}1{Delta}. Therefore, our results indicate that the inhibitory effects of 15d-PGJ2 on TNF-{alpha}–induced VCAM-1 and ICAM-1 expression are mediated by the functions of ROR{alpha}.



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  		Figure 5. Deletion mutant of ROR{alpha}1 (ROR{alpha}1{Delta}) attenuates RORE-dependent transcription activities of ROR{alpha}1 and ROR{alpha}4. CV-1 cells were transiently cotransfected with the pTK-RORE-Luc plasmid (Firefly), pRL-TK (Renilla), and pcDNA or pcDNA encoding ROR{alpha}1, ROR{alpha}4, or ROR{alpha}1{Delta} as indicated. Results are presented as fold induction of the basal activity of pcDNA. *P<0.05; ***P<0.001.



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  		Figure 6. Deletion mutant of ROR{alpha}1 (ROR{alpha}1{Delta}) attenuates the effects of ROR{alpha}1 and 15d-PGJ2 on TNF-{alpha}–induced expression of VCAM-1 (A and C) and ICAM-1 (B and C). HUVECs were exposed to Ad-ROR{alpha}1{Delta} (moi 20), Ad-ROR{alpha}1 (moi 10), or Ad-lacZ (moi 10, 20, or 30) for 6 hours. The moi of each group was totally adjusted to 30 by addition of Ad-lacZ. After treatment with 15d-PGJ2 (3 µmol/L) or DMSO for 22 hours, cells were stimulated with TNF-{alpha} (0.2 ng/mL) for 2 hours. Quantitative real-time RT-PCR was performed (A and B), and Western blot analysis was performed (C) as described. The normalized expression of VCAM-1 or ICAM-1 to GAPDH is presented as the relative expression to the basal expression in nonstimulated cells. *P<0.05; **P<0.01.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we demonstrate that 15d-PGJ2 induced ROR{alpha}1 and ROR{alpha}4 expression and inhibited TNF-{alpha}–induced VCAM-1 and ICAM-1 expression in HUVECs. In addition, we found that the dominant-negative form of ROR{alpha}1 attenuated the effect of 15d-PGJ2 on the TNF-{alpha}–induced gene expression, suggesting the anti-inflammatory actions of 15d-PGJ2 may be mediated in part by the functions of ROR{alpha}.

15d-PGJ2 is emerging as a key anti-inflammatory mediator.10–14 The anti-inflammatory actions of 15d-PGJ2 have been considered to be mediated through its interaction with PPAR{gamma}. The effects of 15d-PGJ2 on iNOS promoter activity, NF-{kappa}B activity, and AP-1 activity in RAW 264.7 cells, which do not express PPAR{gamma}, were obtained after transient expression of PPAR{gamma}.14 In contrast, in a number of studies, high-affinity synthetic PPAR{gamma} ligands such as rosiglitazone required higher concentrations to obtain anti-inflammatory actions than those necessary for the same effect by 15d-PGJ2.10,14 Furthermore, in the absence of PPAR{gamma} expression or in the presence of a RRPA{gamma} antagonist such as BADGE, 15d-PGJ2 has been shown to regulate NF-{kappa}B, AP-1, or NFAT signaling pathways.14,17,18 These studies suggest that the effects of 15d-PGJ2 on proinflammatory gene expression are mediated through a PPAR{gamma}-independent pathway. Our results in this report suggest that the anti-inflammatory properties of 15d-PGJ2 might be, in part, via ROR{alpha}, which could then inhibit the NF-{kappa}B signaling pathway.29,30

Two lines of evidence in our study suggest that there are 2 independent signaling pathways in the regulation of ROR{alpha} expression: PPAR{gamma}-dependent and PPAR{gamma}-independent pathways. First, we found that 3 µmol/L 15d-PGJ2 induced ROR{alpha}1 and ROR{alpha}4 expression, whereas 10 µmol/L pioglitazone only induced ROR{alpha}4 expression (Figure 1). In contrast, 3 µmol/L 15d-PGJ2 showed a much lower level of PPAR{gamma} activation than 10 µmol/L pioglitazone (Figure 2), and our results in CV-1 cells were consistent with the previous report on HUVECs.32 Second, BADGE attenuated pioglitazone-induced ROR{alpha}4 expression but did not affect 15d-PGJ2–induced ROR{alpha}1 and ROR{alpha}4 expression (Figure 3). In addition, we found that PGD2, the precursor of 15d-PGJ2, and PGA2, which does not interact with PPAR{gamma}, also induced ROR{alpha} expression in HUVECs (data not shown). Together, these results suggest that ROR{alpha} expression is mediated by PPAR{gamma}-dependent and PPAR{gamma}-independent pathways.

VCAM-1 and ICAM-1 expression are induced with TNF-{alpha}, IL-6, and LPS, mediated in part by the NF-{kappa}B signaling pathway.3–5 15d-PGJ2 inhibits NF-{kappa}B activation after stimulation with IL-6 and LPS as well as TNF-{alpha}.14,17,24,32,33 Jackson et al clearly showed that 15d-PGJ2 inhibited PMA- and LPS-induced VCAM-1 expression.24 Furthermore, ROR{alpha} inhibited NF-{kappa}B activation by LPS as well as TNF-{alpha}.30 These observations suggest that 15d-PGJ2 and ROR{alpha} inhibit steps within the common signal pathways after stimulation of the cells with TNF-{alpha} and LPS, such as the NF-{kappa}B signaling pathway. The mechanisms by which ROR{alpha} inhibits the NF-{kappa}B signaling pathway in ECs are still unclear.29 There are many potential mechanisms that could inhibit the NF-{kappa}B signaling pathway, such as a reduction in I{kappa}B kinase (IKK) activity, inhibition of I{kappa}B degradation, and direct interaction of NF-{kappa}B with ROR{alpha}. In addition, there is the possibility that ROR{alpha} may inhibit downregulation of the TNF receptor. It has been demonstrated recently that 15d-PGJ2 represses NF-{kappa}B activation by inhibiting the IKK complex activity,34 and, in addition, 15d-PGJ2 directly inhibits the DNA binding activity of NF-{kappa}B through alkylation of a cysteine residue located in the DNA binding domain of the p65 subunit.33 Our report does not exclude direct effects of 15d-PGJ2 on IKK activity and NF-{kappa}B activity but expands the mechanisms of PPAR{gamma}-independent actions of 15d-PGJ2 to include ROR{alpha}-dependent actions regulating the NF-{kappa}B signaling pathway.

We demonstrate here that 15d-PGJ2 inhibited TNF-{alpha}–induced VCAM-1 and ICAM-1 expression in ECs. However, Marx et al reported that 15d-PGJ2, troglitazone, as well as rosiglitazone did not affect TNF-{alpha}–induced VCAM-1 and ICAM-1 expression.26 The difference in the concentration of TNF-{alpha} (0.2 ng/mL in our study versus 10 ng/mL) may explain this discrepancy because we found that inhibition of VCAM-1 and ICAM-1 expression by 15d-PGJ2 was less when higher amounts of TNF-{alpha} were used to stimulate the ECs (1 or 10 ng/mL) rather than the concentration used here (data not shown). Pasceri et al reported recently that 15d-PGJ2, but not ciglitazone, inhibits TNF-{alpha}–induced VCAM-1 and ICAM-1 expression in ECs.25 Our results are similar, but the HUVECs were treated with 10 µmol/L 15d-PGJ2 for 2 hours rather than 3 µmol/L for 24 hours. We found that treatment of HUVECs with 10 µmol/L 15d-PGJ2 for 24 hours affected their viability (43%). Consistent with our observation, high concentrations of 15d-PGJ2 have been reported frequently to inhibit cell growth or induce apoptosis,19 and, in particular, 10 µmol/L 15d-PGJ2 has been reported to induce apoptosis of HUVECs with the cell viabilities reported to be between 30% and 70%.36–38 However, lower concentrations of 15d-PGJ2 show cytoprotective effects.38 We found that treatment with 3 µmol/L of 15d-PGJ2 for 2 hours did not significantly affect ROR{alpha} expression (data not shown). Together, our observations support the possibility that there may be at least 2 independent mechanisms by which 15d-PGJ2 regulates TNF-{alpha}–induced VCAM-1 and ICAM-1. One mediated through induced ROR{alpha} expression (long treatment with 15d-PGJ2), and another, PPAR{gamma}-independent pathway, directly inhibiting NF-{kappa}B activity (short treatment with high concentrations of 15d-PGJ2). These pathways may play a role in the effects of 15d-PGJ2 on chronic inflammation and acute inflammation, respectively.

ROR{alpha} is expressed in human SMCs and ECs,28,29 and overexpression of ROR{alpha} suppresses TNF-{alpha}–induced expression of proinflammatory genes.29,30 Staggerer mice, the ROR{alpha} gene of which has a deletion in the ROR{alpha} LBD sequence causing a frame shift in the protein, have an increased susceptibility to atherosclerosis and show alterations in several immune responses.39,40 ROR{alpha} expression was shown recently to be increased in the mouse ischemic hind limb.41 In addition, Besnard et al found that hypoxia induces ROR{alpha} expression in ECs and SMCs.28 These results support the possibility that the expression levels of ROR{alpha}, as well as concentration of its ligands, affect the regulation of inflammatory responses in the vascular wall. Increased ROR{alpha} expression would be involved in a protective role against inflammation and ischemia. In agreement with this possibility, our results show that the deletion mutant of ROR{alpha}1 (ROR{alpha}1{Delta}), expressing a similar protein to that of Staggerer mice,29 missing its transcriptional activity, and blocking transcription mediated by ROR{alpha}1 and ROR{alpha}4 (Figure 5), attenuated the suppressive effect of ROR{alpha}1 on TNF-{alpha}–induced gene expression (Figure 6), suggesting that functional ROR{alpha} affects proinflammatory gene expression. Although 15d-PGJ2 showed a minimal effect on ROR{alpha} expression (2- to 3-fold; Figure 1), the effects of 15d-PGJ2 on TNF-{alpha}–induced gene expression were clearly attenuated by the dominant-negative form of ROR{alpha}1 (Figure 6), suggesting the 15d-PGJ2–induced ROR{alpha} would be biologically meaningful. Therefore, our results imply a novel link between 15d-PGJ2 and ROR{alpha} that protects against inflammation in the vascular wall.

We have tested whether 15d-PGJ2 activated ROR{alpha} by the assay system that measures the RORE-dependent transcriptional activity of ROR{alpha}, but 15d-PGJ2 did not affect the basal transcription activity of ROR{alpha} (data not shown). Recently, the crystal structure of ROR{alpha} LBD was determined42 and, furthermore, cholesterol and its derivatives were shown to be natural ligands of ROR{alpha}.42,43 Therefore, we believe that 15d-PGJ2 did not activate ROR{alpha} directly but regulated ROR{alpha} expression levels, resulting in effects on inflammatory responses.

Delerive et al30 and ourselves29 reported that infection with the adenovirus encoding ROR{alpha} led to overexpression of ROR{alpha} in vascular SMCs and ECs, respectively, and showed that ROR{alpha} protein was overexpressed with the antibody against ROR{alpha}. Unfortunately, we were unable to detect endogenous ROR{alpha} protein expression in ECs and SMCs with this antibody, even though we used concentrated nuclear extracts. Delerive et al also reported that they failed to detect endogenous ROR{alpha} protein in SMCs and suggested that this was because of the low affinity of the antibody. However, we clearly show that 15d-PGJ2 induces ROR{alpha} mRNA expression and that overexpression of a dominant-negative form of ROR{alpha}1 attenuated the effect of 15d-PGJ2 on TNF-{alpha}–induced gene expression, suggesting that the expression and functions of ROR{alpha} are significantly upregulated by 15d-PGJ2.

15d-PGJ2 is physiologically present in body fluids at picomolar to nanomolar concentrations and increased in pathological conditions, such as infection and inflammations.44,45 However, Bell-Parikh et al suggested that in vivo 15d-PGJ2 levels are several orders of magnitude below the levels required to induce many of the biological effects of this molecule, using 3T3-L1 adipocytes.46 Shibata et al demonstrated endogenous production of 15d-PGJ2 in human atherosclerotic lesions and hypothesized that PG is involved in atherosclerotic inflammation.47 In addition, arachidonate metabolism is greatly increased under pathological conditions, such as inflammation, and local concentrations of PGs have been shown to be in the micromolar range.

In conclusion, we demonstrated for the first time that 15d-PGJ2 induced ROR{alpha} expression in ECs and that ROR{alpha} expression was mediated in part by a PPAR{gamma}-independent pathway. We have further characterized the induced ROR{alpha} expression and shown that 15d-PGJ2 inhibited TNF-{alpha}–induced VCAM-1 and ICAM-1 expression dependent on the functions of ROR{alpha}. These findings show that 15d-PGJ2 regulates inflammatory responses independent of PPAR{gamma} in the vascular wall, suggesting ROR{alpha} as a target molecule for regulating vascular inflammation.


*    Acknowledgments
 
We thank Kohich Kawai, Noboru Satozawa, Takashi Sato, Akihiro Oyabe, Masaaki Ishii, Junko Hosoya, and Yuko Horimizu (Nihon Schering) for their support.

Received October 25, 2004; accepted January 7, 2005.


*    References
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