The microRNA-17–92 (miR-17–92) cluster has been associated with the maturation of the immune system and the development of hematopoietic tumors and other malignancies (1–3). The polycistronic primary transcript C13orf25 encodes 6 distinct members of the cluster: miR-17, 18a, 19a, 19b, 20a, and 92a (2, 4). Amplification of miR-17–92 has been observed in malignant diseases, leading to advantages in growth and survival due to the silencing of tumor-suppressor genes, such as cyclin-dependent kinase inhibitor 1A (p21), PTEN, or Bcl-2–interacting protein Bim, by individual miR-17–92 members (2, 3, 5–7). In miR-17–92–knockout mice, skeletal defects during embryogenesis were noted (8), and the animals died directly after birth due to severe developmental defects of the lung and the heart (9). In contrast, moderate overexpression of miR-17–92 in the lymphoid lineage in mice was found to lead to lymphoproliferative disease and autoimmunity (5). Interestingly, the expression of miR-17–92 is triggered by inflammatory cytokines, such as interleukin-6 (IL-6) (10, 11).
Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads ultimately to the destruction of joints and bones. The cause of RA is still unknown, but it is clear that the disease is driven by infiltrating immune cells and by resident cells within the joint, as well as the mutual interaction between these cells (12). In this regard, RA synovial fibroblasts (RASFs) have been identified as key players in disease pathogenesis. Within the inflamed synovium, RASFs are stimulated by cytokines and other molecules to activate major signaling pathways, which leads to overproduction and secretion of matrix-degrading enzymes and mediators of inflammation. Immune cells are attracted to the synovium and are activated by RASF-secreted cytokines, and in turn, they stimulate RASFs, resulting in a self-sustaining cycle of chronic joint inflammation (13). Due to the disturbed expression pattern of various protooncogenes and tumor suppressors, the aggressive phenotype of the RASFs has been compared with the behavior of a locally invading tumor (14), and as such, these cells promote adhesion to, and destruction of, articular cartilage (12, 15). Similar to the pathogenesis of malignant tumors, aberrant expression of several miRNAs has recently been associated with the pathogenesis of RA (16).
MicroRNAs are characterized by their ability to target different messenger RNAs (mRNAs) simultaneously, which might influence several signaling pathways at once. MicroRNAs are considered to act by fine-tuning gene expression. The effects of a single miRNA on a specific target might thus be rather weak; however, by repressing many targets at the same time, a significant alteration of gene expression can occur (17–18).
We have previously shown that IL-6 induces miR-17–92, both in pulmonary arterial endothelial cells and in human hepatocytes, enhancing the acute-phase response in the latter cells (10, 11). Thus, to further elucidate the role of miR-17–92 in inflammatory processes, we examined the expression and function of miR-17–92, in particular, miR-18a, in RASFs in relationship to tumor necrosis factor α (TNFα), one of the major proinflammatory cytokines involved in the pathogenesis of RA (19, 20).
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In this study, we have shown that the miR-17–92 cluster is induced in RASFs by TNFα through activation of NF-κB and that miR-18a is involved in the up-regulation of both constitutive and TNFα-induced secretion of MMP-1 and inflammatory cytokines and chemokines and, thus, increases the chemoattractive potential of RASFs. Moreover, using reporter gene assays, we identified TNFAIP-3 as a novel direct target of miR-18a, and we demonstrated that by repressing TNFAIP-3 expression, miR-18a enhances NF-κB signaling in RASFs. Thus, miR-18a constitutes a novel positive feedback loop of the NF-κB signaling pathway via translational repression of TNFAIP-3 (Figure 6D), which might further aggravate the activated phenotype of synovial fibroblasts in the pathogenesis of RA.
In recent years, it has become clear that miRNAs are dysregulated in RA and that, acting as repressors of gene expression, they contribute to autoimmunity and joint destruction (for review, see ref.16). The miR-17–92 cluster has been associated with important inflammatory processes, such as the acute-phase response (11), and maturation of the immune system. Xiao and colleagues (5), for example, have shown that in mice, overexpression of miR-17–92 in lymphocytes led to the development of autoimmunity, as reflected by the production of autoantibodies and the infiltration of lymphocytic cells into nonlymphoid tissues. In a mouse model of systemic lupus erythematosus, miR-17–92 was found to be specifically up-regulated in splenic T cells (32), and another study showed that miR-17 and miR-19b are critical regulators of Th1 cell responses and Treg cell differentiation (33).
We found in our study an up-regulation of the miR-17–92 primary transcript and its mature miRNAs in response to TNFα, which in turn, repressed the NF-κB pathway inhibitor TNFAIP-3, thus mediating proinflammatory functions in our experiments. A recent study by Philippe et al (34), on the other hand, found miR-19a and miR-19b to be repressed in RASFs by stimulation with Toll-like receptor 2 (TLR-2) and TLR-4 ligands. Moreover, TLR-2 was identified as a direct target of the miR-19 family (34). Thus, it may be assumed that different signals connected to the pathogenesis of RA might lead to differential expression and function of miR-17–92 in RASFs. Interestingly, it has been predicted that miR-19a, similar to miR-18a, targets TNFAIP-3 (www.targetscan.org), and our own unpublished data using reporter gene assays with the 3′-UTR of TNFAIP-3 confirmed this prediction. Furthermore, miR-19 was shown to repress the NF-κB inhibitor CYLD (35). Gantier and colleagues (36) very recently found that miR-19b targets not only TNFAIP-3, but also other negative regulators of NF-κB signaling. Based on these findings, we strongly propose a proinflammatory role of miR-17–92 in the pathogenesis of RA.
In our screening to detect a functional role of miR-17–92 in RASFs, we chose MMP-1 expression as readout and found that miR-18a had significant effects on mRNA levels of MMP-1. Further analysis showed that miR-18a also enhanced the expression of other important mediators involved in the pathogenesis of RA, including IL-6, IL-8, MCP-1, and RANTES. These findings, together with data from our previous work showing that miR-18a targets the protein inhibitor of activated STAT-3 (PIAS-3), an inhibitor of STAT-3 signaling (11), we postulated that miR-18a might similarly target inhibitors of TNFα-induced signaling pathways, and we found by a computational approach using TargetScan and subsequent validation experiments, that miR-18a acts as a repressor of TNFAIP-3. Interestingly, it has been shown that knockout of TNFAIP-3 in myeloid cells triggers the development of an erosive polyarthritis (37); conversely, adenoviral delivery of TNFAIP-3 was shown to improve inflammation and bone destruction in collagen-induced arthritis (25). These data show that the NF-κB signaling pathway is a crucial mediator of arthritis, and they imply that controlling NF-κB activity (directly via TNFAIP-3 or indirectly through miR-18a) in the different cell types involved in the pathogenesis of RA may be a promising therapeutic approach.
In addition to TNFAIP-3, we identified PTP4A3 as a new target of miR-18a by use of a reporter gene assay. However, transfection with pre-miR-18a did not reduce PTP4A3 levels in RASFs. In general, target repression by miRNAs through the miRNA-induced silencing complex may be additionally regulated by RNA binding proteins that facilitate or inhibit the interaction of miRNA with mRNA, depending on different cellular and environmental conditions (38). One might speculate that the functional discrepancy observed between 3′-UTR targeting of PTP4A3 by miR-18a in HEK 293 cells and in RASFs result from the different cellular environments. It thus appears that repression of TNFAIP-3 is the major contributor to the miR-18a–mediated up-regulation of MMP-1 and inflammatory cytokines.
Silencing of TNFAIP-3, however, did not entirely duplicate the effects of miR-18a transfection, as shown strikingly by experiments involving IL-8. MicroRNA-18a increased the expression of IL-8 only when cells were stimulated with TNFα, whereas silencing of TNFAIP-3 additionally up-regulated the constitutive expression of IL-8 mRNA. These results indicate that additional, as-yet-unidentified, targets of miR-18a may be responsible for the final gene expression observed after pre-miR-18a transfection. One such target may be connective tissue growth factor, which was shown to be de-repressed by anti–miR-18a treatment in glioblastoma spheroid cultures (39) and which has been connected to the expression of IL-6 and IL-8 in tendon fibroblasts (40). The involvement of connective tissue growth factor in MMP-1 and cytokine expression in RASFs remains elusive for the moment and warrants further experimental analysis.
This study is the first to show that miR-18a is part of a positive regulatory loop in NF-κB signaling in RASFs. Studies from recent years have established a complex network of miRNAs that positively and negatively regulate NF-κB signaling at different levels of the signaling cascade (for review, see ref.41). Two miRNAs that have been associated with the pathogenesis of RA are miR-146a and miR-155, the latter of which is involved in the production of proinflammatory cytokines and the development of collagen-induced arthritis (42–45). Both of them are induced through NF-κB–dependent pathways but seem to be engaged in negative feedback loops. MicroRNA-155, for example, may repress inflammatory signaling by targeting the inhibitor of κB kinases β and ε (46). Together, these data support the importance of intact NF-κB signaling. Different levels of regulation appear to include the involvement of various miRNAs to result in a finely balanced activation pattern that becomes disturbed under pathologic conditions such as RA.
In conclusion, the results of this study, together with our previous data (11), underscore the role of miR-18a as a regulator of intracellular signaling pathways that acts as an endogenous amplifier of extracellular signals. More work is needed to complete the picture of miR-18a functions in cell signaling and to further unravel the role of miR-17–92 in autoimmune diseases, particularly in RA. Nevertheless, we have shown that miR-18a is induced by TNFα and is part of a positive feedback loop in the NF-κB signaling pathway in RASFs. MicroRNA-18a thus increases the expression of MMP-1 and cytokines and enhances the chemoattractive potential of RASFs by potentiating the effect of TNFα. These data suggest that miR-17–92 plays an important role in TNFα-mediated signaling mechanisms, which although not directly evidenced by our experiments, might result in RASF-mediated cartilage destruction and immune cell infiltration of the joint in the pathogenesis of RA.
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- MATERIALS AND METHODS
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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. Trenkmann 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. Trenkmann, Brock, R. E. Gay, Michel, S. Gay, Huber.
Acquisition of data. Trenkmann, Brock.
Analysis and interpretation of data. Trenkmann, Brock, S. Gay, Huber.