Activated rheumatoid arthritis synovial fibroblasts (RASFs) play an initiating role in driving RA (1). The inflammatory response is regulated by cytokines and reactive oxygen species (ROS) produced by synoviocytes (2, 3). Levels of proinflammatory cytokines such as interleukin-1β (IL-1β) are elevated in the RA synovium and induce the expression of inflammatory genes and mediators such as the eicosanoids via various signaling pathways (3). The generation of eicosanoids such as prostaglandin E2 (PGE2) is initiated by phospholipase A2 (PLA2) and inducible cyclooxygenase 2 (COX-2) (4–7). Mice deficient in cytosolic PLA2α (cPLA2α) have been reported to reduce eicosanoid production in the presence of collagen-induced arthritis, indicating that cPLA2 plays a critical role in the pathogenesis of RA (8).
ROS have been implicated in inflammatory responses through the activation of transcription factors NF-κB and activator protein 1 (AP-1) (9, 10) and signaling pathways such as MAPKs, leading to the expression of proinflammatory genes in various tissues (11–13). NADPH oxidase (NOX) is a major source of ROS production under various pathologic conditions. The NOX complex is composed of 2 membrane-located subunits p22phox and gp91phox, cytosolic proteins p47phox and p67phox, and a GTPase Rac1, which assemble at membrane sites upon cell activation. Upon exposure to cytokines, synovial NOXs produce superoxide anions, which activate multiple signaling pathways, leading to the expression of inflammatory genes (3, 11). We have therefore suggested that excessive ROS generation appears to be one of the major mediators in the pathogenesis of RA.
Activation of NOX has been shown to stimulate the phosphorylation of p47phox by protein kinase C (PKC) or MAPKs, which initiates assembly of the cytoplasmic components and translocation to the membrane in various cell types (14–16). Production of ROS by hyperoxia was also shown to be mediated through activation of NOX and to be regulated by p42/p44 MAPK and p38 MAPK in human pulmonary artery endothelial cells (17). NOX-dependent ROS generation and activation of NF-κB and AP-1 have been shown to induce cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) (12). It has also been shown that the H2O2-induced increase in the release of arachidonic acid and the production of PGE2 through the up-regulation of cPLA2 and COX-2 was mediated through Ca2+/PKC/MAPKs and epidermal growth factor receptor transactivation in mouse embryonic stem cells (18). However, very little is known about whether p47phox phosphorylation, NOX activation, and ROS generation are involved in IL-1β–induced cPLA2 expression in RASFs.
Heme oxygenase 1 (HO-1) is a stress-response protein involved in various inflammatory disorders (19, 20). Down-regulation of HO-1 is associated with increased inflammation and oxidative stress in various tissues (21). Induction of HO-1 attenuates the IL-1β–induced expression of matrix metalloproteinases 1 and 3 in osteoarthritic synoviocytes (22). Overexpression of HO-1 has also been shown to suppress the tumor necrosis factor α (TNFα)–mediated expression of inflammatory mediators through attenuation of ROS production in HTSMCs (20). However, it is still unknown whether the IL-1β–induced cPLA2 expression in RASFs is modulated by HO-1.
We therefore performed experiments in human RASFs and in mice to investigate whether HO-1 regulates IL-1β–induced cPLA2 expression. Our findings suggest that in RASFs, IL-1β stimulates the activation of p42/p44 MAPK and JNK-1/2, which leads to NOX-dependent ROS production, AP-1 activation, and cPLA2 gene expression. Overexpression of HO-1 could exert protective effects in the pathogenesis of RA via inhibition of these signaling components.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In RA, inflammation of the synovium largely contributes to the development of the disease symptoms and the tissue degradation. Cytosolic PLA2 is considered to play a pivotal role in eicosanoid production, which is implicated in the pathogenesis of collagen-induced arthritis in mice (8). Moreover, cPLA2 activity gradually increases and shows a correlation with severity of arthritis (4). Although these findings have suggested that cPLA2 may be a causative factor in inflammation and tissue injury in collagen-induced arthritis, the molecular mechanisms underlying the IL-1β–induced expression of cPLA2 in RASFs are still unclear. In this study, we found that IL-1β–induced cPLA2 expression was mediated through activation of p42/p44 MAPK– and JNK-1/2–dependent NOX/ROS generation, leading to activation of AP-1 in RASFs (Figure 6J). Moreover, HO-1 exerts antiinflammatory and antioxidative effects on various cell types (20, 31, 32). We found that overexpression of HO-1 attenuated IL-1β–induced cPLA2 expression, which was mediated through the down-regulation of p47phox-dependent ROS production, leading to abrogation of AP-1 activation and cPLA2 expression in RASFs.
ROS are implicated in the pathogenesis of a wide variety of human inflammatory diseases, such as RA (11). In human RASFs, IL-1β induces the formation of intracellular oxidants, which lead to augmented expression of genes for matrix-degrading enzymes (33). One of the major sources of ROS are the nonphagocytic NOX isoforms, which also play a role in the regulation of signaling cascades in various cell types, including endothelial cells and cardiomyocytes (34). Two different NOX homologs (NOX-2 and NOX-4) were recently identified in RA cells. In synoviocytes, NOX-4 could be responsible for a continuous superoxide production and NOX-2 could be responsible for superoxide production by cytokines (3). We also confirmed in the present study that NOX-2 and NOX-4 are expressed in RASFs, as determined by reverse transcription–PCR (data not shown). Moreover, the expression of cPLA2 was shown to be due to NOX activation and ROS generation by cigarette smoke extract in HTSMCs (12). We therefore investigated the roles of the NOX/ROS cascade associated with cPLA2 expression by IL-1β in RASFs. The use of NOX inhibitors or siRNAs significantly abolished the IL-1β–induced cPLA2 expression, suggesting that NOX-2 is essential for these responses. The results are consistent with those of a previous study showing that IL-1β induces ROS production in RA cells, which is inhibited by NOX inhibitors (3). These results suggested that NOX-2–dependent ROS generation may play a key role in IL-1β–induced cPLA2 expression in RASFs.
Production of superoxide anions by NOX is accompanied by extensive phosphorylation of p47phox, which is crucial for translocation of the cytosolic components and assembly of the active NOX (14, 16). Activation of NOX-2 is initiated by the assembly of p47phox with gp91phox. In vascular smooth muscle cells, p47phox is phosphorylated on serine and tyrosine residues and translocated from the cytosol to the membrane by angiotensin II (35). TNFα-induced activation of Src kinase in HTSMCs results in tyrosine phosphorylation and translocation of p47phox with increased ROS generation (20). It has been shown that phosphorylation of p47phox on Ser345 is directly related to TNFα-induced ROS production in neutrophils isolated from the synovial fluid of RA patients (36). IL-1β–induced p47phox phosphorylation has also been detected in RA synoviocytes (3). Although agonist-induced p47phox phosphorylation has been extensively investigated in various cell types, the mechanisms of phosphorylation of p47phox and activation of NOX in RASFs are unclear. Our data from the present study provide evidence for the in vitro phosphorylation of p47phox by IL-1β and interaction between NOX-2 and p47phox in the regulation of IL-1β–induced ROS generation. These results suggested that IL-1β induces NOX-2/ROS generation and cPLA2 expression via phosphorylation of p47phox subunit. Moreover, other NOX-2 subunits (p67phox or Rac1) may also be involved in superoxide production induced by cytokines.
MAPKs have been shown to regulate cPLA2 expression in various cell types (37, 38). Our results revealed that IL-1β–stimulated phosphorylation of p42/p44 MAPK and JNK-1/2 was inhibited by their respective inhibitors. Moreover, IL-1β–induced cPLA2 expression was also significantly inhibited by these pharmacologic inhibitors and shRNAs, indicating that p42/p44 MAPK and JNK-1/2 participate in cPLA2 induction by IL-1β in RASFs. These results are consistent with previous reports from studies of HTSMCs (38). Moreover, p42/p44 MAPK and p38 MAPK play a role in the hyperoxia-induced NOX/ROS cascade in human pulmonary artery endothelial cells (17). In vascular smooth muscle cells, angiotensin II activated MAPKs (p42/p44 MAPK, p38 MAPK, and JNK-1/2) in association with NOX-mediated generation of ROS (39). TNFα-stimulated JNK-1/2 phosphorylation led to ROS generation, potentiating the necrosis of fibroblasts (40). Our results indicated that IL-1β induced ROS generation via p42/p44 MAPK and JNK-1/2 in RASFs.
Phosphorylation of p47phox occurs at serine residues, suggesting the potential involvement of different protein kinases, such as PKC or MAPKs, in these responses (39). It has been shown that p42/p44 MAPK activation involves the granulocyte–macrophage colony-stimulating factor–induced phosphorylation of p47phox at Ser345, while p38 MAPK modulates the TNFα-induced phosphorylation of the same site on p47phox in neutrophils isolated from the synovial fluid of RA patients (3). Moreover, we demonstrated a link between the MAPKs, p47phox phosphorylation, and NOX/ROS generation in RASFs in response to IL-1β. These results further suggest that NOX-dependent ROS generation is involved in IL-1β–induced cPLA2 expression via MAPK-mediated p47phox phosphorylation in RASFs.
The transcription factor AP-1 is typically composed of c-Jun and c-Fos proteins and is implicated in the up-regulation of several inflammatory genes, such as cPLA2 (39). Thus, the role of AP-1 in IL-1β–induced cPLA2 expression was revealed by using a selective AP-1 inhibitor and by transfection with siRNA for c-Jun or c-Fos in RASFs. Our results suggested that AP-1 is essential for cPLA2 expression induced by IL-1β. ROS has also been shown to activate NF-κB and AP-1 in HTSMCs (12). Our data showed that IL-1β induced the accumulation of phospho–c-Jun in the nucleus, which was reduced by blockade of the NOX-2/ROS cascade. We further demonstrated that IL-1β stimulated the activity of AP-1 and increased the binding of c-Jun and c-Fos to the AP-1 element within the cPLA2 promoter. In contrast, the IL-1β–induced accumulation of c-Fos in the nucleus and the binding of c-Fos to the cPLA2 promoter were attenuated only by U0126 and NAC. We further confirmed by using an AP-1-mutated cPLA2 construct that the AP-1–binding site (–498 to –492) within the cPLA2 promoter is required for IL-1β–induced cPLA2 transcription activity. These results indicated that IL-1β–induced cPLA2 expression might be mediated through the p42/p44 MAPK, JNK-1/2, and NOX/ROS cascades, leading to activation of AP-1 in RASFs.
Up-regulation of HO-1 serves as an adaptive response to protect cells from stress. The protective effects of HO-1 could be related to generation of the antioxidant molecules and are of particular interest in the context of inflammatory responses (41). Recent studies have revealed that HO-1 induction in animals protects them from the development of arthritis (42). It has been reported that induction of HO-1 by cobalt protoporphyrin IX in the presence of IL-1β decreases the expression of matrix metalloproteinase 1/3 in osteoarthritic synoviocytes (22). HO-1 induction was also shown to attenuate the expression of inflammatory cytokines and COX-2 in RASFs (29). Moreover, overexpression of HO-1 suppressed TNFα-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression by reducing NOX/ROS generation (20).
Our data also showed that HO-1 exerted antiinflammatory effects on IL-1β–induced cPLA2 expression in RASFs. Overexpression of HO-1 attenuated p47phox phosphorylation, NOX activity, and ROS generation. HO-1 induction by Ad-HO-1 also inhibited the formation of p47phox and NOX-2 complexes by IL-1β in RASFs. Several transcription factors have been shown to be redox-sensitive, including AP-1 (2). Our observations confirmed that IL-1β–induced accumulation of phospho–c-June and c-Fos in the nucleus and AP-1 promoter activity are reduced by HO-1 induction. Thus, AP-1 appears to be one of the important targets for the action of HO-1 in RASFs. Here we have provided evidence of the protective role of HO-1 induction against IL-1β–induced cPLA2 expression by the inhibition of NOX/ROS generation in RASFs.
In summary, IL-1β induced ROS generation through NOX activation and, in turn, initiated the activation of AP-1. The activation of NOX in response to IL-1β was probably dependent on p42/p44 MAPK– and JNK-1/2–mediated phosphorylation of p47phox. Activated AP-1 was recruited to the promoter regions of cPLA2, which led to increased cPLA2 promoter activity and the expression of cPLA2 mRNA and protein in RASFs. Moreover, overexpression of HO-1 suppressed p47phox phosphorylation, NOX activation, ROS generation, and AP-1 activation, resulting in the inhibition of cPLA2 expression in RASFs. Therefore, cPLA2 might be an important ROS-sensitive gene that is expressed in RASFs. Inhibition of p47phox phosphorylation by HO-1 induction might be a mechanism that contributes to the tight regulation of NOX activity and thereby diminishes redox-sensitive proteins in inflammation.
- Top of page
- 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. Yang 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. Chi, Y.-W. Chen, Hsiao, Y.-L. Chen, Yang.
Acquisition of data. Chi, Y.-W. Chen, Hsiao, Y.-L. Chen, Yang.
Analysis and interpretation of data. Chi, Y.-W. Chen, Hsiao, Y.-L. Chen, Yang.