To investigate the role of histone H3 lysine 4 (H3K4) methylation in interleukin-1β (IL-1β)–induced cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in human osteoarthritic (OA) chondrocytes.
To investigate the role of histone H3 lysine 4 (H3K4) methylation in interleukin-1β (IL-1β)–induced cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in human osteoarthritic (OA) chondrocytes.
Chondrocytes were stimulated with IL-1, and the expression of iNOS and COX-2 messenger RNA and proteins was evaluated by real-time reverse transcriptase–polymerase chain reaction analysis and Western blotting, respectively. H3K4 methylation and the recruitment of the histone methyltransferases SET-1A and MLL-1 to the iNOS and COX-2 promoters were evaluated using chromatin immunoprecipitation assays. The role of SET-1A was further evaluated using the methyltransferase inhibitor 5′-deoxy-5′-(methylthio)adenosine (MTA) and gene silencing experiments. SET-1A level in cartilage was determined using immunohistochemistry.
The induction of iNOS and COX-2 expression by IL-1 was associated with H3K4 di- and trimethylation at the iNOS and COX-2 promoters. These changes were temporally correlated with the recruitment of the histone methyltransferase SET-1A, suggesting an implication of SET-1A in these modifications. Treatment with MTA inhibited IL-1–induced H3K4 methylation as well as IL-1–induced iNOS and COX-2 expression. Similarly, SET-1A gene silencing with small interfering RNA prevented IL-1–induced H3K4 methylation at the iNOS and COX-2 promoters as well as iNOS and COX-2 expression. Finally, we showed that the level of SET-1A expression was elevated in OA cartilage as compared with normal cartilage.
These results indicate that H3K4 methylation by SET-1A contributes to IL-1–induced iNOS and COX-2 expression and suggest that this pathway could be a potential target for pharmacologic intervention in the treatment of OA and possibly other arthritic diseases.
Osteoarthritis (OA) is the most common form of arthritis and is a leading cause of disability in the elderly (1). Clinical manifestations of OA may include pain, stiffness, and reduced joint motion. Pathologically, OA is characterized by progressive degeneration of articular cartilage, synovial inflammation, and subchondral bone remodeling. These processes are thought to be largely mediated through excess production of proinflammatory and catabolic mediators. Among these mediators, interleukin-1β (IL-1β) has been demonstrated to be predominantly involved in the initiation and progression of the disease (2–4). One mechanism through which IL-1 exerts its effects is by up-regulating the expression of genes encoding for inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and the release of nitric oxide (NO) and prostaglandin E2 (PGE2) (2–4).
The production of NO is an important component in the pathogenesis of OA, and increased levels of nitrite/nitrate have been observed in the synovial fluid and serum of arthritis patients (5). The biosynthesis of NO is catalyzed by a group of enzymes known as NO synthases (NOS). There are 3 distinct NOS. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, while the iNOS is expressed following stimulation with a variety of inflammatory agents, such as endotoxins or cytokines (6). NO participates in the pathogenesis of arthritis by inducing chondrocyte apoptosis (7) and matrix metalloprotease (MMP) production (8) and by suppressing the synthesis of collagen and proteoglycans (9). In addition, NO enhances the production of inflammatory cytokines (5) and PGE2 (10) and reduces the synthesis of endogenous IL-1 receptor antagonist (IL-1Ra) (11). The important role of NO in the pathogenesis of OA is further supported by the finding that selective inhibition of iNOS in an experimental model of OA reduces the structural changes and the expression of several inflammatory and catabolic factors (12).
Like NO, PGE2 contributes to the pathogenesis of arthritis through several mechanisms, including up-regulation of MMP (13) and IL-1 (14) production, enhancement of the degradation of cartilage matrix components (15), and promotion of chondrocyte apoptosis (16). In addition, PGE2 mediates pain responses and potentiates the effects of other mediators of inflammation (17). COX is the key enzyme in the biosynthesis of PGE2, and 2 isoforms have been identified. COX-1 is constitutively expressed in a wide variety of tissues and is responsible for housekeeping functions. In contrast, COX-2 is undetectable in most normal tissues, but is rapidly induced by growth factors and proinflammatory cytokines, such as IL-1 and tumor necrosis factor α (TNFα) (17). COX-2 expression and activity are increased in cartilage from OA patients, and this is thought to play a primary role in the pain and inflammation associated with the disease (18). Moreover, COX-2 inhibitors have been extensively used in the treatment of OA.
Posttranslational modifications of nucleosomal histones, including acetylation, methylation, phosphorylation, and sumoylation, play important roles in the regulation of gene transcription through remodeling of chromatin structure (19, 20). To date, histone acetylation and methylation are among the most studied and best characterized modifications. Unlike acetylation, which is generally associated with transcriptional activation, histone-lysine methylation is associated with either gene activation or repression, depending on the specific residue modified (21–24). For instance, methylation of the histone H3 lysine-4 (H3K4) is commonly associated with transcriptional activation, whereas methylation of H3K9 correlates with transcriptional repression (21–24). In addition, H3K4 can be mono-, di-, or trimethylated, with the di- and trimethylated forms being the most positively correlated with transcriptional activation (21–24).
H3K4 methylation is catalyzed by the action of a family of histone methyltransferases (HMTs) that share a conserved SET domain, which was named for its presence in diverse Drosophila chromatin regulators: Su(var)3–9, Enhancer of Zeste (E[z]) and Trithorax (Trx). Several specific H3K4 methyltransferases have been identified and characterized, including SET-1A, SET-1B, and 4 mixed-lineage leukemia (MLL) family HMTs (MLL-1, MLL-2, MLL-3, and MLL-4). Among them, only SET-1A and MLL-1 are able to di- and trimethylate H3K4 (25–28).
Although the induction of iNOS and COX-2 expression by IL-1 in chondrocytes is well documented (2–4), the role of histone methylation in their regulation remains undefined. In this study, we examined the role of H3K4 methylation in IL-1–induced iNOS and COX-2 expression in chondrocytes.
Recombinant human IL-1 was obtained from Genzyme. Aprotinin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, and 5′-deoxy-5′-(methylthio)adenosine (MTA) were from Sigma-Aldrich Canada. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal calf serum (FCS), and TRIzol reagent were from Invitrogen. Antibodies against iNOS and COX-2 were purchased from Cayman Chemical. Antibody against β-actin was from Santa Cruz Biotechnology. Antibodies against histone H3 and against mono-, di-, and trimethylated H3K4 were from Upstate/Millipore. Anti–SET-1A and anti–MLL-1 antibodies were from Bethyl Laboratories. Polyclonal rabbit anti-mouse IgG coupled with horseradish peroxidase (HRP) and polyclonal goat anti-rabbit IgG coupled with HRP were from Pierce.
Normal human cartilage (from femoral condyles) was obtained at necropsy, within 12 hours of death, from donors who had no history of arthritic diseases (n = 14; mean ± SD age 59 ± 13 years). To ensure that only normal tissue was used, cartilage specimens were thoroughly examined both macroscopically and microscopically. Only those found to be free of alterations by both methods were further processed. OA cartilage was obtained from patients undergoing total knee replacement surgery (n = 48; mean ± SD age 63 ± 19 years). All OA patients were diagnosed with knee OA according to the criteria developed by the American College of Rheumatology (29). At the time of surgery, the patients had symptomatic disease requiring medical treatment in the form of nonsteroidal 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. Informed consent was obtained from each donor or from an authorized third party.
Chondrocytes were released from cartilage by sequential enzymatic digestion, as previously described (30). Cells were seeded at 3.5 × 105/well in 12-well culture plates (Costar) or at 6–7 × 105/well in 6-well culture plates in DMEM supplemented with 10% FCS, and cultivated at 37°C for 48 hours. Cells were washed and incubated for an additional 24 hours in DMEM containing 0.5% FCS before stimulation with IL-1.
Histones were extracted from the cells as previously described (31). Briefly, cells were washed with phosphate buffered saline (PBS) and lysed with ice-cold lysis buffer containing 10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 1.5 mM PMSF, 1 mM Na3VO4, and 10 μg/ml of aprotinin, leupeptin, and pepstatin. Sulfuric acid was added to a concentration of 0.2N, and the resultant supernatant was collected and dialyzed twice against 0.1M acetic acid and 3 times against sterile water.
Whole-cell lysates were prepared and analyzed as previously described (30).
Total RNA from stimulated chondrocytes was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To remove contaminating DNA, the isolated RNA was treated with RNase-free DNase I (Ambion). The RNA was quantitated using the RiboGreen RNA quantitation kit (Molecular Probes), dissolved in diethylpyrocarbonate-treated-H2O and stored at –80°C until used. One microgram of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Fermentas) as detailed in the manufacturer's guidelines. One-fiftieth of the RT reaction was analyzed by real-time PCR as described below. The following primers were used: for iNOS, 5′-ACATTGATGAGAAGCTGTCCCAC-3′ (sense) and 5′-CAAAGGCTGTGAGTCCTGCAC-3′ (antisense); for COX-2, 5′-TGTGTTGACATCCAGATCAC-3′ (sense) and 5′-ACATCATGTTTGAGCCCTGG-3′ (antisense); and for GAPDH, 5′-CAGAACATCATCCCTGCCTCT-3′ (sense) and 5′-GCTTGACAAAGTGGTCGTTGAG -3′ (antisense).
Real-time PCR analysis was performed in a total volume of 50 μl containing template DNA, 200 nM sense and antisense primers, 25 μl of SYBR Green Master Mix (Qiagen), and 0.5 units of uracil N-glycosylase (UNG; Epicentre Technologies). After incubation at 50°C for 2 minutes (UNG reaction), and at 95°C for 10 minutes (UNG inactivation and activation of the AmpliTaq Gold enzyme), the mixtures were subjected to 40 amplification cycles (15 seconds at 95°C for denaturation and 1 minute for annealing and extension at 60°C). Incorporation of SYBR Green dye into the PCR products was monitored in real time using a GeneAmp 5700 Sequence detection system (Applied Biosystems), allowing determination of the threshold cycle (Ct), at which exponential amplification of PCR products begins. After PCR, dissociation curves were generated with 1 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).
Relative messenger RNA (mRNA) expression in chondrocytes was determined 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 (unstimulated cells) from the ΔCt value of each treatment. Fold changes compared with the control 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 for each independent experiment.
The ChIP experiments were performed according to the ChIP protocol provided by Upstate/Millipore and previously published protocols (32, 33). The primer sequences used were as follows: for the iNOS promoter, 5′-ATGAACTGCCACCTTGGACT-3′ (sense) and 5′-GTTTTCGACTCGCTACAAAGTT-3′ (antisense); for the COX-2 promoter, 5′-AAGACATCTGGCGGAAACC-3′ (sense) and 5′-ACAATTGGTCGCTAACCGAG-3′ (antisense); and for the MMP-13 promoter, 5′-ATTTTGCCAGATGGGTTTTG-3′ (sense) and 5′-CTGGGGACTGTTGTCTTTCC-3′ (antisense).
Specific small interfering RNA (siRNA) for SET-1A, MLL-1, or scrambled control was obtained from Dharmacon. Chondrocytes were seeded in 6-well plates at 6 × 105 cells/well and incubated for 24 hours. Cells were transfected with 100 nM siRNA using HiPerFect Transfection Reagent (Qiagen) following the manufacturer's recommendations. The medium was changed 24 hours later, and the cells were incubated for an additional 24 hours before stimulation with 100 pg/ml of IL-1 for 2 hours or 20 hours.
Cartilage specimens were processed for immunohistochemistry as previously described (30). The specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) of paraffin-embedded specimens were deparaffinized in toluene and dehydrated in a graded series of ethanol. The specimens were then preincubated with chondroitinase ABC (0.25 units/ml in PBS, pH 8.0) for 60 minutes at 37°C, followed by a 30-minute incubation with 0.3% Triton X-100 at room temperature. Slides were then washed in PBS followed by 2% hydrogen peroxide/methanol for 15 minutes. They were further incubated for 60 minutes with 2% normal serum (Vector) and overlaid with primary antibody for 18 hours at 4°C in a humidified chamber. The antibody was a rabbit polyclonal anti-human SET-1A (Bethyl Laboratories), which was used at 10 μg/ml.
Each slide was washed 3 times in PBS, pH 7.4, and stained using the avidin–biotin complex method (Vectastain ABC kit; Vector). The color was developed with 3,3′-diaminobenzidine (Vector) containing hydrogen peroxide. The slides were counterstained with eosin. The specificity of staining was evaluated by using antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of the protein fragment corresponding to amino acids 1200–1250 of human SET-1A (Bethyl), and by substituting nonimmune rabbit IgG (Chemicon) for the primary antibody at the same concentration. The evaluation of positive-staining chondrocytes was performed using our previously published method (30). For each specimen, 6 microscopic fields were examined under 40× magnification. The total number of chondrocytes and the number of chondrocytes staining positive were evaluated, and the results were expressed as the percentage of chondrocytes staining positive (cell score).
Results of the real-time PCR and ChIP analyses are expressed as the mean ± SD, and statistical significance was assessed by Student's 2-tailed t-test. Results of the immunohistochemical analyses are expressed as the median (range), and statistical analysis was performed using the nonparametric Mann-Whitney U test. P values less than 0.05 were considered statistically significant.
We first examined the effect of IL-1 on iNOS and COX-2 mRNA expression in human OA chondrocytes. Cells were stimulated with IL-1 for various time periods, and the levels of iNOS and COX-2 mRNA were determined by real-time RT-PCR. IL-1–induced changes in gene expression were expressed as the fold change over control (untreated cells) after normalization to the internal control GAPDH. Treatment with IL-1 (100 pg/ml) induced iNOS mRNA expression in a time-dependent manner. Levels of mRNA for iNOS started to gradually increase at 2 hours after stimulation to reach a peak at 6 hours. With the longer incubation times, we observed a gradual decline in the mRNA levels starting at 8 hours. Similarly, treatment with IL-1 led to a time-dependent increase in COX-2 mRNA (data available upon request from the author). COX-2 mRNA was rapidly and significantly induced at 1 hour following stimulation with IL-1, reached the maximum at 6 hours and started to decrease at 8 hours (data available upon request from the author).
Next, we performed Western blot analysis to determine whether changes in mRNA levels were paralleled by changes in iNOS and COX-2 protein levels. Consistent with its effects on iNOS and COX-2 mRNA, IL-1 induced the expression of iNOS and COX-2 protein in a time-dependent manner (data available upon request from the author). By 4 hours poststimulation, iNOS protein levels were significantly increased. These levels were further increased up to 8 hours and remained elevated until 24 hours. The induction of COX-2 protein expression occurred earlier (2 hours poststimulation) than iNOS protein expression, reached the maximum at 8 hours, and remained constant until 24 hours (data available upon request from the corresponding author). These results confirmed that IL-1 is a potent inducer of iNOS and COX-2 expression in chondrocytes (2–4).
Recent studies have provided abundant evidence indicating that histone methylation plays an important role in the regulation of gene expression and that H3K4 di- or trimethylation is strongly correlated with transcriptional activation when found at promoter sites (21–24). To determine whether H3K4 methylation might be involved in IL-1–induced COX-2 and iNOS transcription, we performed ChIP assays. Chondrocytes were stimulated with IL-1 for various time periods, and formaldehyde cross-linked DNA–proteins were immunoprecipitated using antibodies specific for mono-, di-, or trimethylated H3K4. Control Ig and no antibodies were used as controls. DNA isolated from the immunoprecipitates was analyzed by real-time PCR using specific primers spanning the transcription start site (+1), the TATA box, and the binding sites of several transcription factors in the proximal regions of the iNOS (bp –256 to +24), COX-2 (bp –270 to +7), and MMP-13 (bp –220 to + 7) promoters.
As shown in Figures 1A–C, treatment with IL-1 enhanced the levels of di- and trimethylated H3K4 at the iNOS and COX-2 promoters in a time-dependent manner. In contrast, the levels of H3K4 methylation at the MMP-13 promoter remained unchanged, indicating that the observed modifications at the iNOS and COX-2 promoters are specific. The levels of di- and trimethylated H3K4 at the iNOS and COX-2 promoters were significantly increased at 0.5 hours after IL-1 stimulation, reached a maximum at 1–2 hours, and returned to a near basal level by 8 hours, whereas the level of monomethylated H3K4 did not appreciably change following IL-1 stimulation (Figure 1A). No immunoprecipitable COX-2 or iNOS promoter DNA was detected with the control Ig and with the no antibodies controls (data not shown). The induction of H3K4 di- and trimethylation by IL-1 at the iNOS and COX-2 promoter paralleled the increased transcription of iNOS and COX-2 (data available upon request from the author), suggesting that enhanced H3K4 di- and trimethylation may play a key role in IL-1–induced iNOS and COX-2 expression.
To determine whether the changes in H3K4 methylation seen at the iNOS and COX-2 promoters were not secondary to events causing global H3K4 methylation, we investigated the effect of IL-1 on global H3K4 methylation in chondrocytes. Cells were stimulated with IL-1 for various time periods, histones were extracted, and the levels of H3K4 methylation were measured by Western blot analysis using specific antibodies for mono-, di-, or trimethylated H3K4. As shown in Figure 1, the levels of mono-, di-, or trimethylated H3K4 were high in untreated chondrocytes, and treatment with IL-1 did not significantly change these levels. These results indicate that the alterations in H3K4 methylation seen in the ChIP assays were not due to nonspecific global histone modifications and are specific for the iNOS and COX-2 promoters.
SET-1A and MLL-1 are H3K4-specific methyltransferases capable of di- and trimethylating H3K4 (25–28). Hence, we performed ChIP assays in IL-1–treated chondrocytes to examine the recruitment of SET-1A and MLL-1 to the iNOS and COX-2 promoters. As shown in Figure 2A, treatment with IL-1 resulted in sustained recruitment of SET-1A at the promoters of iNOS and COX-2. In contrast, IL-1 had no effect on the recruitment of MLL-1 to either promoter (Figure 2B), suggesting that the H3K4 methyltransferase that is involved in H3K4 methylation at the iNOS and COX-2 promoters is SET-1A. No immunoprecipitable COX-2 or iNOS promoter DNA was detected with the control Ig and no antibodies controls (data not shown). Strikingly, SET-1A was recruited to the promoters of iNOS and COX-2 when the levels of di- and trimethylated H3K4 increased (Figures 1B and C), and this recruitment correlated well with the increased transcription of iNOS and COX-2 (data available upon request from the author). Immunoblotting of cell lysates did not show any changes in the levels of SET-1A protein (Figures 2C and D), suggesting that the enhanced recruitment of SET-1A to the iNOS and COX-2 promoters seen with the ChIP assays was not due to increased expression of SET-1A protein. Together, these data suggest an implication of SET-1A in IL-1–induced H3K4 methylation and iNOS and COX-2 expression.
The previous data suggest that SET-1A is involved in H3K4 di- and trimethylation and may contribute to the induction of iNOS and COX-2 expression. To test this, we first investigated the effect of MTA, a histone methyltransferase inhibitor (34), on IL-1–induced H3K4 methylation at the iNOS and COX-2 promoters. Chondrocytes were pretreated with increasing concentrations of MTA for 1 hour, before stimulation with IL-1 for an additional 1.5 hours. The status of H3K4 methylation at the iNOS and COX-2 promoters was evaluated using ChIP assays with antibodies against mono-, di-, and trimethylated H3K4. We found that MTA treatment dose-dependently decreased IL-1–induced di- and tri-methylation of H3K4 (Figures 3B and C), which had increased during transcriptional activation. However, MTA treatment did not change the level of H3K4 monomethylation, which was not affected during transcriptional activation of iNOS and COX-2 (Figure 3A).
Next, we investigated the effect of MTA on IL-1–induced iNOS and COX-2 protein expression. Chondrocytes were pretreated with increasing concentrations of MTA for 1 hour, before stimulation with IL-1 for 20 hours. As shown in Figure 3D, treatment with MTA dose-dependently suppressed the IL-1–induced iNOS and COX-2 expression. This reduction was coincident with the decline in H3K4 methylation following treatment with MTA. The inhibition observed was not a result of reduced cell viability, as confirmed by MTT assay (data not shown). These findings strongly suggest that the SET-1A methyltransferase activity contributes to IL-1–induced H3K4 methylation at the iNOS and COX-2 promoters as well as iNOS and COX-2 expression.
To confirm the role of SET-1A, we examined the impact of its silencing by siRNA on IL-1–induced H3K4 di- and trimethylation at the iNOS and COX-2 promoters. Chondrocytes were transfected with the scrambled control siRNA, siRNA for SET-1A, or siRNA for MLL-1, and after 48 hours of transfection, the cells were stimulated or were not stimulated with IL-1 for 1.5 hours. SET-1A knockdown reduced IL-1–induced H3K4 di- and trimethylation at the iNOS and COX-2 promoters (Figure 4A). In contrast, MLL-1 silencing had no effect (Figure 4B). These results support the notion that SET-1A mediates IL-1–induced H3K4 di- and trimethylation at the iNOS and COX-2 promoters.
Moreover, SET-1A silencing also markedly suppressed IL-1–induced iNOS and COX-2 expression (Figure 5A), whereas MLL-1 knockdown did not affect iNOS and COX-2 expression (Figure 5B). Taken together, these data strongly suggest that SET-1A contributes to IL-1–induced iNOS and COX-2 expression through up-regulation of H3K4 di- and trimethylation.
To determine whether SET-1A levels were altered under conditions of OA, we performed immunohistochemical analysis on cartilage sections from OA patients and normal donors. As shown in Figures 6A and B, the immunostaining for SET-1A was located in the superficial and upper intermediate zones. Statistical evaluation of the cell score revealed a significant increase in the number of chondrocytes staining positive for SET-1A in OA cartilage (n = 14) as compared with normal cartilage (n = 14). The specificity of the staining was confirmed using an antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of the protein fragment corresponding to amino acids 1200–1250 of human SET-1A (Figure 6C) or nonimmune control IgG (data not shown).
The present study is the first to show that the induction of iNOS and COX-2 expression by IL-1 is accompanied by increased H3K4 di- and trimethylation at the iNOS and COX-2 promoters. These modifications correlated with the recruitment of SET-1A to the iNOS and COX-2 promoters. Blocking methyltransferase activity or reducing the expression level of SET-1A abrogated IL-1–induced H3K4 methylation, as well as iNOS and COX-2 expression. Taken together, these results indicate that H3K4 methylation by SET-1A participates in IL-1–induced iNOS and COX-2 expression and suggest that this pathway may represent a therapeutic target in OA.
Our finding that IL-1–induced transcriptional activation of iNOS and COX-2 is associated with H3K4 di- and trimethylation is consistent with recent studies showing that transcriptional activation of a number of inducible inflammatory genes correlates with increased methylation of H3K4 at target promoters. For instance, the induction of monocyte chemotactic protein 1 (MCP-1) and TNFα by the proinflammatory astrocyte-derived protein S100B or TNFα in THP-1 cells is strongly associated with H3K4 methylation (35). Similarly, H3K4 methylation was reported to be increased at the promoters of TNFα and iNOS upon stimulation of the murine macrophage cell line RAW 264.7 and Kupffer cells with lipopolysaccharide (36). Increased methylation of H3K4 was also observed at promoters of MMP-1 in phorbol 12-myristate 13-acetate–treated T98G cells (31), IL-6 and MCP-1 in TNFα-treated vascular smooth cells (37), class II major histocompatibility complex in IFNγ-treated colon 26 cells (38), and IL-17 in CD4+ T helper cells treated with a combination of transforming growth factor β1 and IL-6 (39).
Several histone methyltransferases have been identified, among which SET-1A and MLL play dominant roles in the di- and trimethylation of H3K4 (25–28). Therefore, we examined the effect of IL-1 on the recruitment of SET-1A and MLL-1 to the iNOS and COX-2 promoters. ChIP results demonstrated that IL-1 enhanced the recruitment of SET-1A to the iNOS and COX-2 promoters, whereas the level of MLL-1 was not affected. Interestingly, the recruitment of SET-1A to the iNOS and COX-2 promoters was concomitant with the appearance of di- and trimethylated H3K4 at these sites, indicating that H3K4 methylation in response to IL-1 could be mediated by SET-1A. It is noteworthy that SET-1A appeared to be maintained at the iNOS and COX-2 promoters when the levels of di- and tri-methylated H3K4 decreased. This suggests that specific H3K4 demethylases or inhibitors of SET-1A activity are recruited to the iNOS and COX-2 promoters and contribute to decreased H3K4 di- and tri-methylation.
The correlation between SET-1A recruitment and H3K4 di- and trimethylation suggests that SET-1A is implicated in these modifications and that H3K4 methylation by SET-1A contributes to IL-1–induced iNOS and COX-2 expression. Indeed, we found that MTA, a protein methyltransferase inhibitor (34), prevented IL-1–induced H3K4 methylation at the iNOS and COX-2 promoters and suppressed IL-1–induced iNOS and COX-2 protein expression. Moreover, the siRNA-mediated knockdown of SET-1A diminished the IL-1–induced di- and trimethylation of H3K4 and blocked the expression of iNOS and COX-2. Collectively, these results suggest that SET-1A contributes to IL-1–induced iNOS and COX-2 expression by enhancing H3K4 methylation.
In addition to H3K4, methylation of H3K9, H3K27, H3K36, and H3K79 is also known to modulate gene transcription. Like H3K4, methylation of H3K36 and H3K79 is associated with transcriptional activation, whereas methylation of H3K9 and H3K27 is associated with transcriptional repression (21–24). Although the role of these modifications in the effects of IL-1 is still unknown, we cannot exclude the possibility that they may also be involved in iNOS and COX-2 transcription.
We also demonstrated that the levels of SET-1A were increased in OA cartilage as compared with normal cartilage. Interestingly, OA chondrocytes in these zones were shown to express elevated levels of iNOS and COX-2 (15, 40, 41). These data, together with the implication of SET-1A in the transcriptional activation of iNOS and COX-2 in cultured chondrocytes, suggest that increased expression of SET-1 may be among the mechanisms that mediate the up-regulation of iNOS and COX-2 OA cartilage.
There are a number of mechanisms by which H3K4 methylation could mediate the transcriptional activation of iNOS and COX-2. One possibility is that H3K4 methylation promotes transcriptional activation by enhancing the acetylation of neighboring histones by histone acetyltransferases and by preventing the binding of the NuRD deacetylase complex (42, 43). Alternatively, methylated H3K4 may serve as a docking site for the recruitment of chromatin-remodeling complexes such as the nucleosome remodeling factor (44), and the chromo–ATPase/helicase–DNA binding domain 1 (45). Finally, H3K4 methylation can activate transcription by facilitating the assembly of active transcription complexes. Indeed, the basal transcription complex TFIID can directly bind to the trimethylated H3K4 via the plant homeodomain finger of its subunit TAF-3 (46), and the methyltransferase SET-1A was reported to associate with RNA polymerase II (47).
In addition to histones, nonhistone proteins, especially transcription factors, have been identified as targets for methylation (48). In this context, Yang et al (49) reported that methylation of the RelA subunit of NF-κB, which is critically involved in the induction of iNOS and COX-2 in chondrocytes, by the lysine methyltransferase SET-7/9 inhibits NF-κB activity by inducing the degradation of RelA. On the other hand, Li et al (35) reported that SET-7/9 associates with the NF-κB p65 and up-regulates the expression of a subset of NF-κB target genes. Whether methylation of NF-κB contributes to the transcriptional activation of iNOS and COX-2 genes in chondrocytes remains to be determined.
In conclusion, the present study provides, to our knowledge, the first evidence that H3K4 methylation by SET-1A contributes to the induction of iNOS and COX-2 expression by IL-1. SET-1A may therefore be a novel therapeutic target for osteoarthritis and other human conditions associated with increased expression of iNOS and COX-2.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Fahmi had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. El Mansouri, Fahmi.
Acquisition of data. El Mansouri, Chabane, Zayed, Fahmi.
Analysis and interpretation of data. El Mansouri, Chabane, Zayed, Kapoor, Benderdour, Martel-Pelletier, Pelletier, Duval, Fahmi.
The authors thank Virginia Wallis for assistance with the manuscript preparation.