Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and variable degrees of bone and cartilage erosion (). Although the etiology and pathogenesis of RA are still poorly understood, accumulating evidence indicates that fibroblast-like synoviocytes (FLS) are important players in all aspects of the pathogenesis of RA ([1-3]). We previously demonstrated that the expression of Cyr61, a secreted extracellular matrix (ECM) protein produced by FLS, is stimulated by interleukin-17 (IL-17), and the overexpressed Cyr61 in turn acts to promote FLS proliferation in an autocrine/paracrine manner, thus contributing to the hyperplasia of synovial lining cells (). Interestingly, Cyr61 can also stimulate FLS to produce IL-6, thus promoting Th17 cell differentiation (). These results not only revealed for the first time that Cyr61 contributes to hyperplasia of synovial lining cells but also established a novel “feed-forward and malicious cycle” that leads to mutual stimulation of FLS and Th17 cells, 2 important cell populations in RA, via Cyr61 overexpression. Our results suggest that in patients with RA, overexpressed Cyr61 is an important mediator in this malicious cycle ([4, 5]). Thus, exploring the mechanisms underlying the dysregulation of Cyr61 expression would contribute to our understanding of the molecular pathogenesis of RA.
The tumor-suppressor protein p53 plays a central role in cell cycle regulation, DNA repair, and apoptosis, and mutations in this gene contribute to the pathogenesis of many neoplastic diseases (). Previous studies have demonstrated overexpression of p53 in RA synovial tissue (). Somatic mutations in the p53 gene have also been observed in RA synovium and in the complementary DNA and genomic DNA obtained from cultured RA FLS, although quantitative differences in mutation frequencies among different studies have been noted ([8, 9]). Furthermore, certain p53 mutations in RA are dominant negative and can suppress the function of endogenous wild-type (WT) p53 (). Inactivation of protein p53 can recapitulate many of the phenotypic changes observed in RA, such as increased proliferation, local IL-6 expression, and invasion of synovial cells ([11-13]). Because RA synoviocytes are a major source of Cyr61 ([4, 5]), inhibiting synovial hyperplasia may reduce Cyr61 production. Nevertheless, whether the somatic mutations of p53 observed in RA synovium are functionally linked to Cyr61 overexpression in RA synovial tissue remains unknown.
Posttranscriptional regulation is commonly used to control messenger RNA (mRNA) degradation and gene expression (). MicroRNAs (miRNAs) are noncoding single-stranded RNAs of 19–23 nucleotides that modulate the expression of multiple proteins at the posttranscriptional level ([15, 16]). MicroRNAs have been demonstrated to have various physiologic and pathologic functions, including regulation of immune-mediated inflammatory disorders ([16, 17]). Recently, dysregulated expression of a dozen miRNAs was observed in patients with RA, in both the circulation and the inflamed synovium (). A previous study showed that miRNA-155 (miR-155) contributed to preeclampsia by down-regulating Cyr61 expression (), and a recent study showed that miRNA–mRNA interaction networks identified targets associated with susceptibility/resistance to collagen-induced arthritis (). However, the question of whether dysregulated expression of miRNAs contributes to the overexpression of Cyr61 in RA synovial tissue has not yet been explored.
MiR-22 is a 22-nucleotide noncoding RNA and was originally identified in HeLa cells as a tumor-suppressing miRNA. Subsequently, miR-22 was found to be ubiquitously expressed in a variety of tissues (). Recent studies showed that miR-22 targets ERα () and ErbB-3 (), promotes hepatitis B virus–related hepatocellular carcinoma development (), and suppresses lung cancer progression by inducing cellular senescence (). Interestingly, p53 was recently shown to engage miRNAs for tumor suppression by inducing the transcriptional expression or maturation of specific miRNAs (). Furthermore, a previous study showed that in the setting of tumor development, p53 signaling might promote miR-22 expression ().
Although previous studies have implicated a role of somatic p53 mutations in RA synovium (), the molecular etiology of p53 mutations in RA has remained largely unknown. In this study, we observed that miR-22 directly targets the 3′-untranslated region (3′-UTR) of Cyr61 and inhibits Cyr61 expression. Furthermore, we demonstrated that the expression of miR-22 was down-regulated and negatively correlated with Cyr61 expression in RA synovial tissue. We further showed that WT p53 positively regulated miR-22 transcription by binding to the promoter region of the miR-22 gene, while mutant p53 found in RA synovial tissue failed to do so. As a result, miR-22 expression is reduced in RA synovial tissue, which contributes to the overexpression of Cyr61 at the posttranscriptional level. Thus, our study revealed that somatic p53 mutations in RA synovial tissue are functionally linked to the inflammation process through a mechanism involving miR-22 and Cyr61.
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CCN1/Cyr61 was the first identified member of the CCN family. Cyr61, a secreted ECM protein, has been shown to regulate angiogenesis as well as cell proliferation, adhesion, migration, and differentiation and is important for wound healing and embryo development ([37-39]). We previously demonstrated that Cyr61 was overexpressed in RA synovial tissue, and that it played a critical role in IL-17–stimulated proliferation of RA FLS (). We further showed that Cyr61 stimulated IL-6 production by FLS via the αvβ5/Akt/NF-κB signaling pathway, which in turn promoted Th17 cell differentiation (). These results not only revealed that Cyr61 contributes to hyperplasia of synovial lining cells but also established a new link between Th17 cells and FLS in RA, whereby the overexpressed Cyr61 acts as a critical mediator in this novel feed-forward and malicious cycle. Nevertheless, whether a posttranscriptional mechanism might underlie the dysregulated expression of Cyr61 in RA remained unclear, nor was it known whether regulation of Cyr61 expression is linked to other RA etiologic factors such as somatic mutations in p53. In this study, we revealed for the first time that a p53/miR-22/Cyr61 axis controls Cyr61 expression in RA FLS.
MiR-22 is encoded within exon 3 of the C17orf91 gene, which is located on chromosome 17p13, a frequently hypermethylated, deleted, or loss of heterozygosity–associated region in cancer ([40, 41]). The 17p13 region also harbors a suite of well-known tumor- suppressor genes, including HCCS1, DPH1, HIC1, XAF1, KCTD11, and P53 ([42-44]). MiR-22 is generally considered to be a tumor suppressor, and several direct target genes of miR-22 have been observed in many cancer types (), but its regulation of Cyr61 was not previously known.
In this study, through a combination of computational prediction and functional analyses, we firmly established that miR-22 negatively regulates Cyr61 expression by directly targeting its 3′-UTR. We further observed that miR-22 was down-regulated in the synovial tissue of patients with RA and was negatively correlated with Cyr61 expression. Further functional studies showed that miR-22 negatively regulated RA FLS proliferation and IL-6 production, and that this effect was mediated by the regulation of Cyr61 levels by miR-22. Taken together, these data reveal a novel miR-22–mediated posttranscriptional mechanism controlling Cyr61 expression and suggest that the decrease in miR-22 levels in RA synovial tissue contributes to Cyr61 overexpression and thus RA pathogenesis.
What mechanism might be responsible for the reduced miR-22 levels in RA synovial tissue? A recent study identified miR-22 as a p53 target gene in the regulation of tumorigenesis (). The p53 protein is a well-known tumor suppressor, and p53 mutations contribute to the pathogenesis of many neoplastic diseases ([6, 36, 45]). Intriguingly, somatic mutations of p53 are frequently observed in RA synovial tissue and are believed to be involved in synovial hyperplasia in RA ([8-12, 32, 33]). Nevertheless, the molecular etiology of p53 mutations in RA was poorly understood. In this study, through a series of experiments, we observed that p53 regulates miR-22 expression in RA FLS at the transcription level through binding to the miR-22 gene locus. Moreover, we conducted a systematic exon sequencing analysis and detected a high frequency of p53 mutations in synovial tissue samples obtained from patients with RA. Additional studies indicated that the mutant forms of p53 in RA synovial tissue are functionally defective, because they failed to activate miR-22 expression. Thus, the somatic mutations of p53 in RA synovial tissue are likely an important reason for the reduced miR-22 levels in RA.
Results of a recent meta-analysis suggested that there was no association between the p53 codon 72 polymorphism and susceptibility to RA (). However, these p53 variants have been shown to influence the function of p53 in apoptosis, cell cycle control, and DNA repair (). We observed that 70.3% of the patients with RA in our study harbored p53 codon 72 mutations, and that the mutant form of p53 is defective in activating miR-22 expression. Thus, our results support the notion that the somatic mutations of p53 observed in RA synovial tissue contribute to disease pathogenesis. Another factor to be considered is IL-6, a key proinflammatory cytokine that is abundant in RA synovium and synovial fluid ([48, 49]) and is known to contribute to RA pathogenesis. Previous studies have shown that p53 may act to repress IL-6 gene expression (), and that p53 mutations in the synovial tissue were associated with increased local expression of IL-6 (). Thus, direct modulation of IL-6 gene expression might be another mechanism by which somatic mutations in p53 could influence RA pathogenesis. Alternatively, dysregulation in the p53/miR-22/Cyr61 axis, as observed in this study, could also contribute to the increase in IL-6 expression, as we have previously shown that Cyr61 can stimulate FLS to produce IL-6 ().
In summary, this study is the first to demonstrate that the involvement of p53 in the posttranscriptional regulation of Cyr61 expression occurs via miR-22. In RA, mutant forms of p53 are unable to activate miR-22 transcription, and the resulting reduction in miR-22 levels leads to enhanced Cyr61 expression, thus contributing to RA pathogenesis. These findings not only provide further insight into the regulation of Cyr61 in RA but also reveal a novel aspect of the molecular etiology of somatic p53 mutations frequently observed in RA synovial tissue.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
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. N. Li 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. Lin, N. Li.
Acquisition of data. Huo, Xiao, Zhu, Xie, S. Sun, He, Shen.
Analysis and interpretation of data. Zhang, Y. Sun, Zhou, Wu, D. Li, N. Li.