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.
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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.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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.