The pathogenesis of rheumatoid arthritis (RA) is characterized by marked proliferation of the synovial tissue, which invades and destroys periarticular bone and cartilage. The major effector cells of cartilage degradation are synovial fibroblasts (1). RA synovial fibroblasts (RASFs) produce high levels of destructive enzymes and proinflammatory cytokines upon activation (2). A characteristic feature of RASFs is their decreased susceptibility to spontaneous as well as FasL- and TRAIL-mediated apoptosis (3, 4). Whereas activation of the Fas pathway is associated with increased JNK signaling, TRAIL has been reported to act via inhibition of the phosphatidylinositol 3-kinase/Akt molecular pathway (5). Several factors have been demonstrated to inhibit receptor-mediated apoptosis in RASFs, such as small ubiquitin-like modifier 1 (6), FLIP (7), and Mcl-1 (8). However, the mechanisms leading to the characteristic apoptosis resistance of RASFs are still incompletely understood.
MicroRNAs (miRNAs) have emerged as fine-tuning regulators of diverse biologic processes (9, 10). During their biogenesis, miRNA genes are transcribed into primary miRNAs that are processed by Drosha and Dicer to generate miRNA duplexes consisting of a mature strand and a passenger miRNA strand (also called miRNA star [miRNA*] strand) (11). Some miRNAs have been reported to be important regulators of cellular lifespan. Of these, miRNA-34a (miR-34a) has been identified as a modulator of apoptosis. However, existing data are controversial. On the one hand, miR-34a was shown to be an inducer of programmed cell death in colon cancer cell lines and mouse embryonic fibroblasts (12–14), whereas on the other hand, miR-34a protected cells, such as human B lymphocytes and a mouse tubular cell line, against apoptosis (15, 16).
Since alterations of apoptosis are a characteristic finding in rheumatoid synovium, we have analyzed the expression of the miR-34 family in RASFs. MiR- 34a, miR-34b/b*, and miR-34c/c* were not found to be differentially expressed between RASFs and synovial fibroblasts from patients with noninflammatory osteoarthritis (OA). However, miR-34a*, the passenger strand of miR-34a, was significantly down-regulated in synovial fibroblasts from patients with RA compared to those with OA. We analyzed the effects of miR-34a* on apoptotic cell death and identified X-linked inhibitor of apoptosis protein (XIAP) as a direct target of miR-34a*. Finally, we showed that low levels of miR-34a* correlate with high XIAP expression in synovial fibroblasts.
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The prolonged cellular lifespan is a fundamental characteristic of activated synovial fibroblasts, contributing to chronic inflammation and joint destruction in RA. In the current study we focused on the expression and function of miR-34, a family of microRNAs that is known to influence apoptotic cell death (12, 15, 23–25). Whereas miR-34a, miR-34b/b*, and miR-34c/c* did not show any significant differences in their expression levels, we demonstrated significant down-regulation of the passenger strand miR-34a* in RASFs compared to OASFs.
Although microRNAs have been the subject of much research in recent years, little is yet known about the relevance of miR passenger strands. Some years ago, it was proposed that regulation of the expression and accumulation of miR/miR* duplexes represents a tissue-dependent mechanism (26). There is evidence of active sorting of both miR and miR* species into regulatory complexes, suggesting that miR* strands may have a biologic role (27). For instance, the passenger strand of miR-17 was shown to directly target the p50 subunit of NF-κB in a human cell line for malignant pleural mesothelioma (28). In addition, miR-30* was able to bind and suppress the translation of target genes, indicating that both the mature and the passenger strands are functional (26). Of relevance to the present results, the passenger strand miR-34* has been found to be localized to argonaute 1, a protein that is crucial for miRs to exert their biologic functions (27, 29).
Recently, our group reported alterations in the DNA methylation status in synovial cells from patients with RA versus those with OA (21). In the present study, we demonstrated that the expression of miR-34a and its passenger strand was increased upon demethylation, although statistical significance was reached only for miR-34a*. Consistent with our data, miR-34a expression has been found to be silenced in various cancers due to aberrant CpG methylation (14, 23). In contrast, cytokines such as TNFα and IL-1β, as well as TLR ligands, did not influence the expression of miR-34a*.
Whereas impaired apoptosis of synovial fibroblasts is important for the pathogenesis of RA, little is known about the miR-dependent regulation of apoptosis. The present findings suggest that both miR-34a and miR-34a* are involved in the regulation of apoptotic pathways. We present evidence here that miR-34a* promotes apoptosis in both FasL- and TRAIL-stimulated RASFs, whereas overexpression of the mature strand miR-34a protects cells against FasL-mediated apoptosis but has no effect on TRAIL-induced cell death.
Conflicting data on the effect of miR-34a on apoptosis have been published. In the colon cancer cell line HCT116, miR-34a was found to promote apoptosis (12). Similarly, miR-34a was found to suppress sirtuin 1, ultimately leading to p53-dependent apoptosis in cancer cells (24). However, a recent study demonstrated antiapoptotic effects of miR-34a in B lymphoid cells (15). Consistent with this finding, up-regulation of miR-34a was positively associated with longer survival of lung cancer cells (23). Concerning the passenger strand miR-34a*, no data on its effect on apoptosis have been published previously. In this study, we demonstrated opposite effects on apoptotic cell death of miR-34a and miR-34a*, with the passenger strand increasing FasL- and TRAIL-induced apoptosis of RASFs. A similar conflicting outcome was observed with miR-155 and its corresponding passenger strand in terms of modulation of interferon-α/β expression (30).
Furthermore, we found that the proapoptotic effects of miR-34a* are mediated upon targeting of XIAP. XIAP is a member of the IAP family of apoptosis inhibitors that block apoptosis by direct binding to caspases, leading to inhibition of the execution phase of apoptosis (31). It was recently shown that XIAP is implicated in innate immunity by mediating signaling of nucleotide-binding oligomerization domain–containing proteins 1 and 2 (32, 33). Consistent with our findings, higher expression levels of XIAP were found in synovial tissue from patients with active RA compared with normal subjects (22) and, in addition, XIAP was reported to be overexpressed in synovial fluid from patients with juvenile idiopathic arthritis (34). Induction of XIAP was observed upon stimulation with hematopoietic cytokines, e.g., granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and stem cell factor, in a human leukemia cell line (35).
Herein we present new evidence of a regulatory role of miR-34a* in the expression of XIAP. Basal expression levels of XIAP correlated with constitutive miR-34a* production, and overexpression/inhibition of miR-34a* modulated XIAP production in RASFs. Using reporter gene assays, we showed that miR-34a* directly targets XIAP via binding to its 3′-UTR region. To our knowledge, this is the first report of an effect of the passenger strand miR-34a* on apoptosis. Our data indicate a functional effect of miR* species, which should be taken into account when analyzing the role of miRs in the pathogenesis of RA.
In conclusion, our results indicate that miR-34a* has a proapoptotic role that is mediated by modulation of the expression of XIAP. As miR-34a* is down-regulated in RASFs, unopposed expression of XIAP may contribute importantly to the apoptosis resistance of these cells.
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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. Kyburz 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. Niederer, Ospelt, R. E. Gay, Detmar, S. Gay, Jüngel, Kyburz.
Acquisition of data. Niederer, Trenkmann, Karouzakis, Kolling, Jüngel.
Analysis and interpretation of data. Niederer, Ospelt, Karouzakis, Neidhart, Stanczyk, S. Gay, Jüngel, Kyburz.