Abstract
Objective. Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2. Early growth response factor-1 (Egr-1) is a key transcription factor in the regulation of mPGES-1, and its activity is negatively regulated by the corepressor NGF1-A-binding protein-1 (NAB1). We examined the effects of valproic acid (VA), a histone deacetylase inhibitor, on interleukin 1ß (IL-1ß)-induced mPGES-1 expression in human chondrocytes, and evaluated the roles of Egr-1 and NAB1 in these effects.
Methods. Chondrocytes were stimulated with IL-1 in the absence or presence of VA, and the level of mPGES-1 protein and mRNA expression were evaluated using Western blotting and real-time reverse-transcription polymerase chain reaction (PCR), respectively. mPGES-1 promoter activity was analyzed in transient transfection experiments. Egr-1 and NAB1 recruitment to the mPGES-1 promoter was evaluated using chromatin immunoprecipitation assays. Small interfering RNA (siRNA) approaches were used to silence NAB1 expression.
Results. VA dose-dependently suppressed IL-1-induced mPGES-1 protein and mRNA expression as well as its promoter activation. Treatment with VA did not alter IL-1-induced Egr-1 expression, or its recruitment to the mPGES-1 promoter, but prevented its transcriptional activity. The suppressive effect of VA requires de novo protein synthesis. VA induced the expression of NAB1, and its recruitment to the mPGES-1 promoter, suggesting that NAB1 may mediate the suppressive effect of VA. Indeed, NAB1 silencing with siRNA blocked VA-mediated suppression of IL-1-induced mPGES-1 expression.
Conclusion. VA inhibited IL-1-induced mPGES-1 expression in chondrocytes. The suppressive effect of VA was not due to reduced expression or recruitment of Egr-1 to the mPGES-1 promoter and involved upregulation of NAB1.
- MICROSOMAL PROSTAGLANDIN E SYNTHASE-1
- CHONDROCYTES
- VALPROIC ACID
- NGF1-A-BINDING PROTEIN-1
Prostaglandin E2 (PGE2) plays an important role in the pathophysiology of arthritis, and excessive levels have been reported in serum and synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA)1. PGE2 contributes to the pathogenesis of arthritis by inducing cartilage proteoglycan degradation2, enhancing the activation and production of matrix metalloproteases (MMP)3, and by promoting chondrocyte apoptosis4. PGE2 is also involved in neoangiogenesis and mediates pain responses5.
The biosynthesis of PGE2 from arachidonic acid requires 2 enzymatic activities. Cyclooxygenase (COX) enzymes convert arachidonic acid into PGH2, which is in turn isomerized to PGE2 by PGE synthase (PGES) enzymes. Two isoforms of the COX enzyme, COX-1 and COX-2, have been identified. COX-1 is constitutively expressed in most tissues, whereas COX-2 is inducible by various stimuli, including proinflammatory signals6. At least 3 distinct PGES isoforms have been cloned and characterized, including cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1), and mPGES-27. cPGES is constitutively and ubiquitously expressed and is functionally coupled with COX-1, promoting immediate production of PGE28. In contrast, mPGES-1 is markedly upregulated by inflammatory or mitogenic stimuli and is functionally coupled with COX-2, promoting delayed PGE2 production9. mPGES-2 is constitutively expressed in various cells and tissues and can be coupled with both COX-1 and COX-210. We and others have previously shown that the level of mPGES-1 is elevated in articular tissues from patients with OA and RA and in animal models of arthritis11,12,13,14. Moreover, mPGES-1 deficiency was protective in animal models of chronic inflammation, pain, and arthritis15,16, which implicates mPGES-1 as a potential target for therapeutic intervention in arthritis.
The expression of mPGES-1 is upregulated in several cell types after treatment with proinflammatory stimuli such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and is downregulated by antiinflammatory glucocorticoids9,11,17. Transcriptional induction of mPGES-1 is primarily controlled by the transcription factor early growth response factor-1 (Egr-1)18,19. Like most transcription factors, the activity of Egr-1 is negatively regulated by corepressor proteins. Two corepressors of Egr-1, NGF1-A-binding proteins (NAB)1 and NAB2, have been identified20,21.
Acetylation and deacetylation of histone and nonhistone proteins play a critical role in the control of gene transcription22,23. The acetylation status is determined by interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC). In general, histone acetylation is associated with transcription activation through relaxed chromatin structure, whereas histone deacetylation is associated with transcription repression via chromatin condensation22,23. Recent studies, however, have revealed that transcription activation is not necessarily associated with histone acetylation and that HDAC activity can also activate transcription. For instance, global analysis of gene expression showed that inhibition of HDAC activity results in both induction and repression of gene expression24,25,26. In addition, genome-wide genetic studies with yeast demonstrated clearly that HDAC are required in both transcriptional activation and repression27,28. Finally, inhibition of HDAC activity was reported to repress transcription in several cell types including chondrocytes29,30,31,32,33,34,35,36
We examined the effect of valproic acid (VA), an HDAC inhibitor37, on IL-1-induced mPGES-1 expression in human OA chondrocytes. We showed that VA suppressed IL-1-induced mPGES-1 expression without interfering with the expression or the recruitment of Egr-1 to the mPGES-1 promoter. Further, we provide evidence that the suppressive effect of VA on IL-1-induced mPGES-1 expression involves upregulation of NAB1.
MATERIALS AND METHODS
Reagents and antibodies
Human recombinant (rh) IL-1 was obtained from Genzyme (Cambridge, MA, USA). TNF-α and IL-17 were purchased from R&D Systems (Minneapolis, MN, USA). VA, cycloheximide (CHX) aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma-Aldrich Canada (Oakville, ON, Canada). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, fetal calf serum (FCS), and Trizol reagent were supplied by Invitrogen (Burlington, ON, Canada). Plasmid DNA was prepared using a kit from Qiagen (Mississauga, ON, Canada). FuGene-6 transfection reagent was from Roche Applied Science (Laval, QC, Canada). The luciferase reporter assay system was from Promega (Madison, WI, USA). Anti-mPGES-1 and anti-cPGES antibodies were from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against Egr-1, NAB1, NAB2, and ß-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit anti-mouse immunoglobulin G (IgG) coupled with horseradish peroxidase (HRP) and polyclonal goat anti-rabbit IgG with HRP were from Pierce (Rockford, IL, USA). All other chemicals were purchased from Fisher Scientific or Bio-Rad (Mississauga, ON, Canada).
Chondrocyte isolation and treatment
Articular cartilage samples from femoral condyles and tibial plateaus were obtained from OA patients undergoing total knee replacement (n = 49, mean age 67 ± SD 17 yrs). Informed consent had been obtained from patients with OA for the use of their tissues for research purposes. All OA patients were diagnosed according to the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA38. At the time of surgery, patients had symptomatic disease requiring medical treatment in the form of non-steroidal antiinflammatory drugs or selective COX-2 inhibitors. Patients who had received intraarticular injection of steroids were excluded. The Clinical Research Ethics Committee of Notre-Dame Hospital approved the study protocol and the use of human articular tissues.
Chondrocytes were released from cartilage by sequential enzymatic digestion as described33. Briefly, small pieces of cartilage were incubated with 2 mg/ml pronase for 1 h followed by 1 mg/ml type IV collagenase (Sigma-Aldrich) for 6 h at 37°C in DMEM and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). Digested tissue was briefly centrifuged and the pellet was washed. The isolated chondrocytes were seeded at high density in tissue culture flasks and cultured in DMEM supplemented with 10% heat-inactivated FCS.
Confluent chondrocytes were detached by trypsinization, seeded at 3.5 × 105 cells per well in 12-well culture plates (Costar, Corning, NY, USA) or at 7 × 105 cells per well in 6-well culture plates in DMEM supplemented with 10% FCS, and cultivated at 37°C for 48 h. Cells were washed and incubated for an additional 24 h in DMEM containing 0.5% FCS and pretreated with VA, trichostatin A (TSA), or BA for 30 min, before stimulation with IL-1, TNF-α, or IL-17. In another set of experiments chondrocytes were pretreated for 30 min with CHX, before stimulation with IL-1 or VA. The expression level of mPGES-1 protein was determined 24 h after stimulation, whereas the level of mPGES-1 messenger RNA (mRNA) was determined at 8 hours. Only first-passaged chondrocytes were used.
PGE2 determination
Levels of PGE2 were determined using a PGE2 enzyme immunoassay (EIA; Cayman Chemical). The detection limit and sensitivity was 9 pg/ml. All assays were performed in duplicate.
Protein extraction and Western blot analysis
For histone extraction, chondrocytes were washed with phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer containing 10 mM hydroxyethyl-piperazine ethanesulfonic acid potassium hydroxide (HEPES-KOH), pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1.5 mM PMSF, 1 mM Na3VO4, and 10 μg/ml aprotinin, leupeptin, and pepstatin. Sulfuric acid was added to a concentration of 0.2 N and the resultant supernatant was collected and dialyzed twice against 0.1 M acetic acid and 3 times against sterile water. For whole-cell lysate preparation, chondrocytes were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 μg/ml each of aprotinin, leupeptin and pepstatin, 1% NP-40, 1 mM Na3VO4, and 1 mM NaF). Lysates were sonicated on ice and centrifuged at 12,000 rpm for 15 min. The protein concentration of the supernatant was determined using the bicinchoninic acid method (Pierce). Then 20 μg of total cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane (Bio-Rad). After blocking in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.1% Tween 20, and 5% (wt/vol) nonfat dry milk, blots were incubated overnight at 4°C with the primary antibody and washed with Tris-buffered saline (TBS), pH 7.5, with 0.1% Tween 20. The blots were then incubated with HRP-conjugated secondary antibody (Pierce), washed again, incubated with SuperSignal Ultra Chemiluminescent reagent (Pierce), and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY, USA).
RNA extraction and cDNA synthesis
Total RNA was isolated from chondrocytes using the TRIzol reagent, and dissolved in 20 μl of diethylpyro-carbonate-treated H2O. One microgram of total RNA was treated with RNase-free DNase and reverse-transcribed using Moloney murine leukemia virus reverse transcriptase. One-fifth of the reverse transcriptase reaction was analyzed by real-time PCR as described below. The following primers were used: mPGES-1: sense 5′-GAA GAA GGC CTT TGC CAA C-3′ and antisense 5′-GGA AGA CCA GGA AGT GCA TC-3′; cPGES: sense 5′-GCA AAG TGG TAC GAT CGA AGG-3′ and antisense 5′-TGT CCG TTC TTT TAT GCT TGG-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense 5′-CAG AAC ATC ATC CCT GCC TCT-3′ and antisense 5′-GCT TGA CAA AGT GGT CGT TGA G-3′.
Real-time PCR
Real-time PCR analysis was performed in a total volume of 50 μl containing cDNA template, 200 nM of sense and antisense primers, and 25 μl of SYBR® Green master mix (Qiagen). Incorporation of SYBR Green dye into PCR products was monitored in real time using a GeneAmp 5700 sequence detector (Applied Biosystems, Foster City, CA, USA) allowing determination of the threshold cycle (CT) at which exponential amplification of PCR products begins. After incubation at 95°C for 10 min to activate the AmpliTaq Gold enzyme, the mixtures were subjected to 40 amplification cycles (15 s at 95°C for denaturation and 1 min at 60°C for annealing and extension). After PCR, dissociation curves were generated, with one peak indicating the specificity of the amplification. A CT value was obtained from each amplification curve using the software provided by the manufacturer (Applied Biosystems). Data were expressed as fold-changes relative to control conditions (unstimulated cells) using the ΔΔCT method as detailed in the manufacturer’s guidelines (Applied Biosystems). A ΔCT value was first calculated by subtracting the CT value for the housekeeping gene GAPDH from the CT value for each sample. A ΔΔCT value was then calculated by subtracting the ΔCT value of the control from the ΔCT value of each treatment. Fold-changes compared with the control (unstimulated cells) were then determined by raising 2 to the ΔΔCT power. Each PCR reaction generated only the expected specific amplicon, as shown by the melting-temperature profiles of the final product and by gel electrophoresis of test PCR reactions. Each PCR was performed in triplicate on 2 separate occasions from at least 3 independent experiments.
Transient transfection
The mPGES-1 promoter construct (−538/−28) was provided by Dr. T. Smith (University of California, Los Angeles, CA, USA). Egr-1 expression vector was donated by Dr. Y. Chen (Morehouse School of Medicine, Atlanta, GA, USA). Expression plasmids for NAB1 and NAB2 were provided by Dr. J. Savren (University of Wisconsin, Madison, WI, USA). The ß-galactosidase reporter vector under the control of SV40 promoter (pSV40-ß-gal) was from Promega. Transient transfection experiments were performed using FuGene-6 transfection reagent according to the manufacturer’s recommendation (Roche Applied Science). Briefly, chondrocytes were seeded 24 h prior to transfection at a density of 3 × 105 cells/well in 12-well plates and transiently transfected with 1 μg of the mPGES-1 promoter construct and 0.5 μg of the internal control pSV40-ß-gal. Six hours later, the medium was replaced with DMEM containing 1% FCS. At 1 day after transfection, the cells were treated with IL-1 in the absence or presence of VA for 18 h. In the overexpression experiments, the amount of transfected DNA was kept constant by using the corresponding empty vector. At the end of the indicated treatment, the cells were washed twice in ice-cold PBS and extracts were prepared for luciferase reporter assay. Luciferase activity was normalized for transfection efficiency using the corresponding ß-galactosidase activity.
RNA interference
Small interfering RNA (siRNA) specific for NAB1 and scrambled control were obtained from Dharmacon (Lafayette, CO, USA). Chondrocytes were seeded in 12-well plates at 3 × 105 cells/well and incubated 24 h. Cells were transfected with 100 nM siRNA using the HiPerFect Transfection Reagent (Qiagen) following the manufacturer’s recommendations. The medium was changed 24 h later and the cells were incubated an additional 24 h before stimulation with IL-1 in the absence or presence of VA. Cell lysates were prepared and analyzed for mPGES-1 or NAB1 protein expression by Western blotting.
Chromatin immunoprecipitation (ChIP) assay
The ChIP experiments were performed according to the ChIP protocol provided by Upstate/Millipore Biotechnology and published protocols39,40. The primer sequences used were mPGES-1 promoter sense 5′-CCC GGA GAC TCT CTG CTT C-3′ and antisense 5′-TCA ACT GTG GGT GTG ATC AGC-3′.
Statistical analysis
Data are expressed as the mean ± SD. Statistical significance was assessed by 2-tailed Student t test. P values < 0.05 were considered statistically significant.
RESULTS
VA suppressed IL-1-induced mPGES-1 protein expression
Blocking histone deacetylation with specific inhibitors modulated gene expression in several cell types29,30,31,32,33,34,35,36. To determine whether HDAC inhibitors can modulate PGE2 production and mPGES-1 expression in chondrocytes, cells were stimulated with IL-1 in the absence or presence of increasing concentrations of VA, and the release of PGE2 and the expression of mPGES-1 protein were evaluated by EIA and Western blotting, respectively. As shown in Figure 1A, stimulation with IL-1 dramatically increased PGE2 production and mPGES-1 protein expression. Treatment with VA suppressed IL-1-induced PGE2 release and mPGES-1 protein expression in a dose-dependent manner. In contrast, the expression of cPGES protein was not affected by these treatments. To determine whether VA inhibits HDAC activity in chondrocytes, we examined its effect on the acetylation of histone H3 protein. As shown in Figure 1B, treatment with VA increased histone H3 protein acetylation in a dose-dependent manner. Thus, VA suppressed IL-1-induced mPGES-1 expression and enhanced histone H3 acetylation in chondrocytes. Treatment of chondrocytes by 2 additional HDAC inhibitors, butyric acid (BA) and VA, also suppressed IL-1-induced mPGES-1 expression in a dose-dependent manner (Figure 1C).
To examine whether the inhibitory effect of VA on mPGES-1 expression was specific for IL-1, we assessed its effects on TNF-α- and IL-17-induced mPGES-1 protein expression. Interestingly, VA suppressed the induction of mPGES-1 expression by both TNF-α and IL-17 (Figure 1D), indicating that its effect was not restricted to IL-1. These data indicate that VA can downregulate the induction of mPGES-1 expression in human chondrocytes. The concentrations of VA utilized did not affect chondrocyte viability as judged by the trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (data not shown).
VA prevented IL-1-induced mPGES-1 expression at the transcriptional level
To determine whether the suppressive effect of VA was due to inhibition of mPGES-1 mRNA induction, chondrocytes were stimulated with IL-1 in the absence or presence of increasing concentrations of VA, and the level of mPGES-1 mRNA expression was determined using real-time PCR. The relative expression level of mPGES-1 mRNA was plotted as fold-change over control untreated cells. GAPDH gene expression was used for normalization. As expected, treatment with IL-1 resulted in a marked increase (∼12-fold) of the mPGES-1 mRNA level, but this effect was dose-dependently attenuated in the presence of VA (Figure 2A), suggesting that VA exerts its effects at the transcriptional level. To confirm this, we carried out transient transfection experiments. Chondrocytes were transfected with the human mPGES-1 promoter-luciferase reporter gene and then stimulated with IL-1 in the absence or presence of VA. As shown in Figure 2B, IL-1 induced the luciferase activity of the mPGES-1 promoter and this activation was dose-dependently reduced by VA, consistent with its effect on mPGES-1 mRNA expression. Taken together, these data indicate that the suppressive effect of VA on IL-1-induced mPGES-1 takes place, at least in part, at the transcriptional level.
VA did not target Egr-1 expression and recruitment to the mPGES-1 promoter, but prevented its ability to transactivate the mPGES-1 promoter. The transcription factor Egr-1 plays a key role in the induction of mPGES-1 expression18,19; therefore, we considered whether the inhibition of IL-1-induced mPGES-1 expression by VA was due to prevention of Egr-1 expression. Chondrocytes were incubated with IL-1 in the absence or presence of VA, and cell extracts were prepared and analyzed by Western blotting. As shown in Figure 3A, IL-1 strongly induced the expression of Egr-1, and this effect was not altered in the presence of VA, indicating that the suppressive effect of VA is not due to reduced expression of Egr-1.
To determine whether VA affects the recruitment of Egr-1 to the endogenous mPGES-1 promoter, we performed ChIP assays. Chondrocytes were stimulated with IL-1 in the absence or presence of VA, and formaldehyde cross-linked DNA proteins were immunoprecipitated with an anti-Egr-1 antibody. No-antibody and non-immune serum were used as controls. DNA isolated from the immunoprecipitates was analyzed by real-time PCR using primers amplifying the mPGES-1 promoter region (bp −142 to −37) that harbors Egr-1 binding sites. As shown in Figure 3B, treatment with IL-1 enhanced (3.2-fold) the binding of Egr-1 to the endogenous mPGES-1 promoter. However, IL-1-induced Egr-1 binding was not affected by VA. We failed to detect immunoprecipitable mPGES-1 promoter DNA with the no-antibody and non-immune serum controls (data not shown). Therefore, VA inhibited IL-1-induced mPGES-1 expression by mechanisms independent of, or in addition to, impaired expression or recruitment of Egr-1 to the mPGES-1 promoter.
Next, we investigated the effect of VA on the ability of Egr-1 to transactivate the mPGES-1 promoter. Chondrocytes were cotransfected with the mPGES-1 promoter and an expression vector for Egr-1 and then left untreated or treated with inceasing concentrations of VA. As shown in Figure 3C, overexpression of Egr-1 highly increased the mPGES-1 promoter activity. Interestingly, treatment with VA dose-dependently attenuated Egr-1-mediated activation of the mPGES-1 promoter. Together, these data suggest that VA inhibits mPGES-1 expression by interfering with the Egr-1 transcriptional activity.
VA-mediated inhibition of IL-1-induced mPGES-1 expression requires de novo protein synthesis
To determine whether the inhibitory effect of VA on IL-1-induced mPGES-1 expression requires de novo protein synthesis, we tested the effect of the protein synthesis inhibitor cycloheximide (CHX). Chondrocytes were pretreated with CHX for 30 minutes and stimulated with IL-1 in the absence or presence of VA for 8 hours. The levels of mPGES-1 mRNA were analyzed by real-time PCR. As shown in Figure 4, pretreatment with CHX blocked VA-mediated inhibition of IL-1-induced mPGES-1 expression, suggesting that the suppressive effect of VA was an indirect effect and was dependent on de novo protein synthesis.
NAB1 contributes to the suppression of IL-1-induced mPGES-1 by VA
The ability of CHX to block VA-mediated suppression of Egr-1-induced mPGES-1 expression suggests that VA induces the synthesis of one or more proteins that suppress Egr-1 activity. Possible candidates that may be involved in the suppressive effect of VA are NAB1 and NAB2. NAB1 and NAB2 negatively regulate the activity of Egr-1 and suppress the transcription of Egr-1-dependent target genes20,21.
To determine whether NAB1 and/or NAB2 were involved in the suppressive effect of VA on IL-1-induced mPGES-1 expression, we first examined their ability to repress Egr-1-induced mPGES-1 promoter activation in chondrocytes. Cells were cotransfected with the mPGES-1 promoter and an expression vector for Egr-1 together with increasing concentrations of vectors encoding for NAB1 or NAB2. As shown in Figure 5A, overexpression of Egr-1 greatly increased the mPGES-1 promoter activity. Interestingly, cotransfection with NAB1 or NAB2 dose-dependently reduced Egr-1-mediated activation of the mPGES-1 promoter. Similarly, overexpression of NAB1 or NAB2 dose-dependently abrogated IL-1-mediated activation of the mPGES-1 promoter (Figure 5B). These experiments demonstrated that NAB1 and NAB2 proteins inhibit both Egr-1- and IL-1-mediated activation of the mPGES-1 promoter in chondrocytes.
Next we analyzed the effect of VA on NAB1 and NAB2 expression in chondrocytes. Cells were treated with VA for different time periods, and the expression of NAB1 and NAB2 proteins was evaluated by Western blotting. As illustrated in Figure 6A, VA enhanced NAB1 expression in a time-dependent manner. NAB1 protein expression started to increase 0.5 hours post-stimulation, reached the maximum at 1 hour, and remained elevated until 12 hours. In contrast, VA had no significant effect on the expression levels of NAB2 (Figure 6A).
Transcriptional repression by NAB proteins requires their recruitment to target promoters through interaction with Egr-141,42. Therefore, we examined whether VA promotes NAB1 recruitment to the endogenous mPGES-1 promoter. As shown in Figure 6B, treatment with either VA or IL-1 alone had no effect on the binding of NAB1 to the mPGES-1 promoter. However, the combined treatment of IL-1 and VA induced the recruitment of NAB1 to the mPGES-1 promoter. We did not detect obvious recruitment of NAB2 to the mPGES-1 promoter. Thus, VA induced NAB1 recruitment to the mPGES-1 promoter in the presence of IL-1, suggesting that NAB1 may contribute to the suppressive effect of VA on mPGES-1 expression.
NAB1 silencing with small interfering RNA (siRNA) blocked VA-mediated suppression of IL-1-induced mPGES-1 expression
To confirm the involvement of NAB1 in the observed effect of VA, we evaluated the effect of NAB1 silencing by siRNA on VA-mediated suppression of IL-1-induced mPGES-1 expression. Chondrocytes were transfected with the scrambled control siRNA or siRNA for NAB1 and after 24 hours of transfection, the cells were stimulated with IL-1 in the absence or presence of VA. As shown in Figure 6C, transfection with NAB1 siRNA antagonized the suppressive effect of VA on IL-1-induced mPGES-1 expression, whereas transfection with scrambled control siRNA had no effect. NAB1 protein levels were almost completely suppressed in chondrocytes transfected with NAB1 siRNA compared to cells transfected with scrambled siRNA, confirming NAB1 gene silencing (Figure 6, lower panel).
These results support the notion that upregulation of NAB1 contributes to the suppression of IL-1-induced mPGES-1 expression by VA.
DISCUSSION
In this study, we demonstrate that treatment of human chondrocytes with VA, an HDAC inhibitor, suppressed IL-1-induced mPGES-1 expression at the transcriptional level. The inhibitory effect of VA was not associated with reduced expression of Egr-1 or its recruitment to the mPGES-1 promoter, and requires de novo protein synthesis. In addition, we identify NAB1 as an essential factor that mediates the suppressive effect of VA. To our knowledge, this is the first study to demonstrate that VA suppresses IL-1-induced mPGES-1 expression by upregulating the expression of NAB1.
Recently, a number of studies have demonstrated that HDAC inhibitors modulate inflammatory responses. For instance, HDAC inhibitors including VA reduced lipopolysaccharide-induced production of IL-1, TNF-α, and interferon-γ (IFN-γ) in human peripheral blood mononuclear cells29, and production of TNF-α, IL-6, and reactive oxygen species in neuroglia cultures and primary microglia30,31. HDAC inhibitors have also been reported to suppress IL-12 production in dendritic cells and macrophages32. Further, we and others have reported that they downregulate inducible nitric oxide synthase and COX-2 expression in several cell types, including chondrocytes33. In vivo, HDAC inhibitors dose-dependently reduced the circulating levels of the proinflammatory cytokines TNF-α, IL-1, and IL-6 in an endotoxemia model34. In addition to their antiinflammatory properties, HDAC inhibitors exhibit chondroprotective effects. Indeed, Young, et al35 showed that HDAC inhibitors blocked the induction of several enzymes responsible for cartilage degradation, including MMP-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS)-4, -5 and -9, and prevented cartilage degradation in an explant assay35. Moreover, we recently showed that HDAC inhibitors downregulate IL-1-induced proteoglycan degradation in cartilage explants33. More recently, Grabiec, et al36 reported that HDAC inhibitors suppressed the production of TNF-α and IL-6 by macrophages from patients with RA36. Together these data suggest that HDAC inhibitors may have protective effects in arthritis. Indeed, they inhibit joint swelling, synovial inflammation, and bone and cartilage destruction in autoantibody-mediated arthritis43 and collagen-induced arthritis models44,45. We have extended these observations by showing that VA inhibits IL-1-induced mPGES-1 expression in human chondrocytes. Moreover, the induction of mPGES-1 expression by TNF and IL-17 was also inhibited by VA, suggesting that VA could repress mPGES-1 expression independently of the stimulus. The repressive effect of VA on IL-1-induced mPGES-1 expression occured at the transcriptional level, as determined by real-time RT-PCR analysis and transient transfection assays.
The transcriptional induction of mPGES-1 is primarily controlled by Egr-1 through 2 Egr-1 binding motifs located in the proximal region of the mPGES-1 promoter18,19. Therefore, we examined whether inhibition of Egr-1 expression and/or recruitment to the mPGES-1 promoter could be the mechanism by which VA prevents IL-1-induced mPGES-1 expression. Our results demonstrated that VA did not affect IL-1-induced Egr-1 expression and recruitment to the mPGES-1 promoter, indicating that VA acts at a step downstream of Egr-1 expression and recruitment to the mPGES-1 promoter. Indeed, VA inhibits both Egr-1 and IL-1-mediated activation of the mPGES-1 promoter.
We also demonstrated that inhibition of de novo protein synthesis blocked VA-mediated suppression of IL-1-induced mPGES-1 expression, suggesting that the suppressive effect of VA is not direct, but rather indirect, through transcription of a target gene that suppresses mPGES-1 expression. Potential candidates that may mediate the suppressive effect of VA are the corepressors of Egr-1 activity, namely NAB1 and NAB2, known to suppress the expression of Egr-1-dependent genes20,21. We found that treatment with VA caused a rapid upregulation of NAB1 expression, whereas the level of NAB2 was not affected, suggesting that the upregulation of NAB1 contributes to the suppressive effect of VA on IL-1-induced mPGES-1 expression. Interestingly, NAB1 expression was reported to be upregulated by 2 antiinflammatory agents: glucocorticoids and aspirin-triggered lipoxin analog46,47.
Further, we found using ChIP assays that NAB1 is recruited to the −142/−37 region (which contains 2 Egr-1 binding sites) of the mPGES-1 promoter when the cells are stimulated with the combination of IL-1 and VA, but not with IL-1 or VA alone. This suggests that NAB1 is recruited to the mPGES-1 promoter by Egr-1. Indeed, it has been shown that Egr-1 and NAB1 can directly interact in vitro and in vivo and that the association of Egr-1 with NAB1 is involved in the repression of several Egr-1-dependent genes41,42. Altogether, these results strongly suggest that upregulation of NAB1 expression and its recruitment to the mPGES-1 promoter mediates the suppressive effect of VA on IL-1-induced mPGES-1 expression. This is further supported by the fact that silencing of NAB1 by specific siRNA blocked the downregulation of IL-1-induced mPGES-1 expression by trichostatin A. This study is the first to our knowledge to show that NAB1 expression is upregulated by VA, and that NAB1 is important for the suppressive effect of VA on IL-1-induced mPGES-1 expression.
It should be noted that siRNA-mediated silencing of NAB1 did not completely block the suppressive effect of VA on IL-1-induced mPGES-1 expression, suggesting that additional NAB1-independent mechanisms also contribute to the suppressive effect of VA. Indeed, we recently showed that HDAC4 contributes to mPGES-1 expression in synovial fibroblasts, and that trichostatin A suppresses IL-1-induced mPGES-1 expression by interfering with this enzyme’s activity40. Modulation of Egr-1 activity or expression by HDAC inhibitors is not mutually exclusive; both may operate concomitantly. Further investigation will be needed to elucidate whether VA modulates mPGES-1 expression in chondrocytes by targeting the activity of HDAC4 or other isoforms.
There are a number of potential mechanisms by which VA could increase NAB1 expression. One possibility is that VA activates NAB1 transcription through inhibition of HDAC activity and subsequent histone hyperacetylation at the NAB1 promoter. Indeed, several studies reported that VA can activate transcription by enhancing histone acetylation at target gene promoters48,49,50. Alternatively, VA may induce NAB1 expression through hyperacetylation of transcription factor or signaling molecules involved in NAB1 expression. Of note, HDAC inhibitors including VA have been shown to modulate the expression of a number of genes by increasing the acetylation levels of key transcription factors51,52. Finally, VA can upregulate NAB1 expression through NAB1 mRNA stabilization. Additional molecular and biochemical studies are needed to delineate the mechanisms by which VA modulates NAB1 expression.
Our data show that suppression of IL-1-induced mPGES-1 expression by VA was not due to altered expression or recruitment of Egr-1 to the mPGES-1 promoter, but instead, was likely due to the repression of Egr-1 transcriptional activity through upregulation of NAB1 expression.
Acknowledgment
The authors thank Virginia Wallis for her assistance with manuscript preparation.
Footnotes
-
Supported by the Canadian Institutes of Health Research (CIHR) Grant MOP-84282. N. Chabane and N. Zayed are supported by fellowships from the CIHR Training on Mobility and Posture Deficiencies (MENTOR) and the Fonds de Recherche en Santé du Québec (FRSQ).
- Accepted for publication November 24, 2010.
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- 42.
- 43.
- 44.
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- 46.
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- 48.
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- 50.
- 51.
- 52.