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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Objective

We previously showed that Cyr61 acts to promote fibroblast-like synoviocyte (FLS) proliferation and Th17 cell differentiation, suggesting that Cyr61 plays an important role in mediating the joint inflammation and damage in rheumatoid arthritis (RA). The aim of this study was to investigate whether Cyr61 expression is regulated at the posttranscription level, and if so, how this regulation connects to other etiologic factors in RA.

Methods

Expression of microRNA-22 (miR-22) in synovial tissue was detected by real-time polymerase chain reaction (PCR) using miRNA-specific TaqMan MGB probes. MicroRNA-22 promoter activity was analyzed using a Dual-Luciferase Reporter Assay. Cytokine expression was measured by enzyme-linked immunosorbent assay, and the expression of other factors was measured by real-time PCR or Western blotting.

Results

MicroRNA-22 directly targeted the 3′-untranslated region of Cyr61 messenger RNA and inhibited Cyr61 expression. Expression of miR-22 was down-regulated and was negatively correlated with Cyr61 expression in RA synovial tissue. Furthermore, wild-type p53 activated miR-22 transcription by binding to the promoter region of the miR-22 gene, while the mutant forms of p53 frequently found in RA synovial tissue were shown to have lost the ability to activate miR-22 expression. As a result, miR-22 was down-regulated, contributing to the overexpression of Cyr61 in RA FLS.

Conclusion

Our results not only reveal a novel mechanism whereby p53 is involved in the posttranscriptional regulation of Cyr61 expression via miRNA-22, but also provide a molecular explanation for the role of somatic mutations of p53, which are frequently observed in RA synovial tissue, in the etiology of this autoimmune disease.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and variable degrees of bone and cartilage erosion ([1]). 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 ([4]). Interestingly, Cyr61 can also stimulate FLS to produce IL-6, thus promoting Th17 cell differentiation ([5]). 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 ([6]). Previous studies have demonstrated overexpression of p53 in RA synovial tissue ([7]). 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 ([10]). 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 ([14]). 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 ([18]). A previous study showed that miRNA-155 (miR-155) contributed to preeclampsia by down-regulating Cyr61 expression ([19]), and a recent study showed that miRNA–mRNA interaction networks identified targets associated with susceptibility/resistance to collagen-induced arthritis ([20]). 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 ([21]). Recent studies showed that miR-22 targets ERα ([22]) and ErbB-3 ([23]), promotes hepatitis B virus–related hepatocellular carcinoma development ([24]), and suppresses lung cancer progression by inducing cellular senescence ([25]). Interestingly, p53 was recently shown to engage miRNAs for tumor suppression by inducing the transcriptional expression or maturation of specific miRNAs ([26]). Furthermore, a previous study showed that in the setting of tumor development, p53 signaling might promote miR-22 expression ([27]).

Although previous studies have implicated a role of somatic p53 mutations in RA synovium ([28]), 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.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Patients and specimens

A total of 48 patients with RA (6 men and 42 women, mean ± SD age 55 ± 9 years [range 38–72 years]) were included in the study. The mean ± SD disease duration of these patients was 15 ± 8 years. The diagnosis of RA fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism criteria for RA ([29]). The control group comprised 30 patients with a diagnosis of osteoarthritis (OA) based on the criteria proposed by Altman et al ([30]). Synovial tissue specimens were obtained from the patients, and FLS were cultured and identified as reported previously ([4]). The demographic and critical clinical characteristics of the patients with RA are shown in Supplementary Table 1 (available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract). All study protocols and consent forms were approved by the Institutional Medical Ethics Review Board of the Shanghai Jiaotong University School of Medicine.

Cell lines

Human skin fibroblast Hs27, HeLa, MDA-MB-231, and HCT 116 cell lines were obtained from ATCC.

Oligonucleotides and plasmids

We used the following miRNA and small interfering RNA (siRNA) oligonucleotides: miR-22 mimics; miR-22 harboring a mutated seed region; p53-targeting small interfering RNA (siRNA) that targeted human TP53 transcript; and a negative control RNA duplex for both miRNA and siRNA (all from Genepharma). The sequence-specific miR-22 inhibitor and its control inhibitor were obtained from Genepharma. All oligonucleotide sequences are shown in Supplementary Table 2 (available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract).

MiR-22 promoter regions were amplified from human genomic DNA (HeLa cells), and the PCR products were cloned into promoter vector pGL3-Basic (Promega) using Kpn I/Xho I and Xho I/Hind III restriction sites to generate miR-22 1.0K, miR-22 1.2K, miR-22 1.3K, and miR-22 3.4K plasmids. The WT p53 coding sequence region was amplified from HCT 116 complementary DNA, and the PCR product was cloned into pEGFP-N3 vector (Promega) using Bgl II/Sal I restriction sites to generate WT p53 plasmid. The Cyr61 3′-UTR region was amplified from MDA-MB-231 genomic DNA, and the PCR product was cloned into siCheck2 using Xho I/Not I restriction sites to generate Cyr61 3′-UTR WT and deletion plasmids. All of the primers and restriction sites are shown in Supplementary Table 2. The mutant p53 plasmids p53 m1, p53 m2, p53 m3, and p53 m4, and the Cyr61 3′-UTR mutant plasmid were prepared at Sangon Biotech.

DNA sequencing

DNA sequencing for p53 exons 2–11 in RA synovial tissue was performed at Sangon Biotech. All of the primers used are shown in Supplementary Table 2.

Real-time PCR analysis

Total RNA was extracted from cells, and real-time PCR was performed as previously described ([5]). The primers used in this study are shown in Supplementary Table 2. Mature miRNAs were detected by TaqMan MicroRNA Assays using looped-primer reverse transcription (RT)–PCR. The miRNA-specific RT primers and the miRNA-specific TaqMan MGB probes for miR-22, miR-34a, miR-133a, miR-146b, miR-155, miR-181a, miR-181c, miR-206, miR-597, and miR-770-5p were obtained from Applied Biosystems and performed according to the manufacturer's protocol.

Western blot analysis

Protein immunoblotting was performed as described previously ([5]). Briefly, cells were transfected with p53 plasmids or RNA oligonucleotides, and the cell lysates or RA and OA synovium lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore) overnight at 30V. The expression of Cyr61 (Santa Cruz Biotechnology), p53, and Akt and the activation of Akt were analyzed using specific antibodies (Cell Signaling Technology). After washing with phosphate buffered saline (PBS), the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG at room temperature for 1 hour, followed by washing with PBS. The target proteins were examined with a Millipore enhanced chemiluminescence system and visualized with autoradiography film. The intensity of the Western blot bands was analyzed by ImageJ software (http://rsb.info.nih.gov/ij).

Cell transfection and luciferase reporter assay

Oligonucleotides or plasmids were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Luciferase reporter assays were performed to characterize the miR-22 promoter and the miR-22–targeted 3′-UTR. The pRL-TK vector, which expresses Renilla luciferase, was cotransfected to correct the differences in both transfection and harvest efficiencies. Cell lysate was collected 48 hours after transfection, and luciferase activities were measured using a Promega Dual-Luciferase Reporter Assay System. The activity of the firefly luciferase reporter was normalized to that of the Renilla luciferase.

Immunofluorescence staining

Frozen cryosections of synovial tissue from patients with RA were stained for immunologic assessment of p53 and Cyr61 expression. Rabbit anti-human Cyr61 (Santa Cruz Biotechnology) conjugated with Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen) and mouse anti-human p53 (Cell Signaling Technology) conjugated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) were used. DAPI (Fluka) was used to stain nuclei. Isotype controls were used for all assays. Immunostainings were analyzed with a fluorescence microscope (Zeiss Axioskop 2 Plus).

Chromatin immunoprecipitation (ChIP) assay

A ChIP assay was performed with a commercially available kit (Beyotime), according to the manufacturer's instructions. Briefly, cells (2 × 106/immunoprecipitation) were crosslinked in 1% formaldehyde for 10 minutes at room temperature, and the crosslink was halted with 0.125M glycine. The primary antibody, p53 rabbit IgG (Cell Signaling Technology), was used to capture the chromatin. After overnight capture at 4°C, chromatin was collected, purified, and de-crosslinked at 65°C. The precipitated DNA fragments were quantified by PCR analysis, using the primer sets shown in Supplementary Table 2.

FLS division assay

FLS were labeled with carboxyfluorescein succinimidyl ester, and the cells were transfected with miR-22, anti–miR-22, or control. After 48 hours, cell division was detected by flow cytometry and analyzed with ModFit LT version 3.3.

Enzyme-linked immunosorbent assay (ELISA).

RA FLS were transfected with miR-22 or anti–miR-22; cell culture supernatants were collected and diluted for the measurement of IL-6, IL-1β, and transforming growth factor β by ELISA (R&D Systems) according to the manufacturer's recommendations. A standard curve method was performed for each plate and used to calculate the absolute concentrations of the indicated cytokines.

Statistical analysis

All experiments were performed in triplicate. Analysis of variance was performed to determined differences among groups, and Student's t-test was used to analyze the differences between 2 groups, using GraphPad Prism version 4.0. The correlation between p21, Bax and Cyr61, and miR-22 and Cyr61 was processed using Microsoft Excel. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

MiR-22 targets the 3′-UTR of Cyr61 mRNA

To identify candidate miRNAs controlling Cyr61 expression, we used the computational program TargetScan to screen for putative miRNAs that might target the 3′-UTR of Cyr61 mRNA. Based on bioinformatics predictions and the conservation pattern observed in mammals, miR-22 emerged as a leading candidate. As shown in Figure 1A, the putative miR-22 target site in the 3′-UTR of Cyr61 is evolutionarily conserved. We thus hypothesized that miR-22 might directly target the 3′-UTR of Cyr61. Indeed, miR-22 inhibited the activity of firefly luciferase reporter containing the 3′-UTR of Cyr61 but not that containing the UTR with the miR-22 target site mutated or deleted (Figure 1B). In contrast, mutant miR-22 that harbored a mutated seed region displayed no effect on reporter activity (Figure 1B). Furthermore, transfection of miR-22 or anti–miR-22 into RA FLS showed that miR-22 regulated the expression of endogenous Cyr61 protein (Figure 1C). Taken together, these results indicated that miR-22 acts to inhibit Cyr61 expression by directly binding to its 3′-UTR.

image

Figure 1. MicroRNA-22 (miR-22) targets the 3′-untranslated region (3′-UTR) of Cyr61. A, Schematic representation of the putative target site for miR-22 in the 3′-UTR of Cyr61. B, Luciferase reporter activity in human skin fibroblasts transfected with wild-type (WT), mutant (MUT), or target-deleted (DEL) 3′-UTR of Cyr61 and negative control (NC). Bars show the mean ± SD of 3 independent experiments. ∗∗ = P < 0.01. C, Western blots showing Cyr61 protein expression in fibroblast-like synoviocytes transfected with miR-22, anti–miR-22, or negative control. Hsa = homo sapiens; Ptr = Pantroglodytes; Mml = Macaca mulatta; Mmu = Mus muscularis. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract.

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MiR-22 is down-regulated in RA synovial tissue and is negatively correlated with Cyr61 expression

Considering that miR-22 inhibits Cyr61 expression and that Cyr61 is overexpressed in RA synovial tissue, it is likely that miR-22 expression may be dysregulated in RA. We therefore examined the expression profile of miR-22 in the synovial tissue of patients with OA and patients with RA, using miRNA-specific TaqMan MGB probes. The results showed that the expression of miR-22 was down-regulated (Figure 2A), while the expression of Cyr61 was increased (Figure 2B), in the synovial tissue of patients with RA. Furthermore, correlation analyses showed a significant negative correlation between the expression level of Cyr61 and that of miR-22 (Figure 2C). These data suggested that decreased miR-22 expression may contribute to increased expression of Cyr61 in patients with RA.

image

Figure 2. MicroRNA-22 (miR-22) expression is down-regulated in rheumatoid arthritis (RA) synovial tissue. A and B, Expression of miR-22 (A) and Cyr61 (B) in osteoarthritis (OA) synovial tissue and RA synovial tissue, as detected by real-time polymerase chain reaction. Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01. C, Negative correlation between the expression of miR-22 mRNA and Cyr61 mRNA (P < 0.0001).

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MiR-22 negatively regulates FLS proliferation and IL-6 production through targeting Cyr61.

Our group previously demonstrated that Cyr61 overexpression in RA FLS enhanced FLS proliferation and promoted Th17 cell differentiation by inducing IL-6 production ([4, 5]). Thus, we sought to determine whether the decrease in miR-22 expression in RA synovial tissue contributes to promoting FLS proliferation and IL-6 production, and whether it does so via a mechanism involving Cyr61. To this end, we investigated the effect of miR-22 on the proliferation and IL-6 production of FLS derived from patients with RA. We transfected the RA FLS with mimetic miR-22 and observed that both FLS proliferation (Figure 3A) and IL-6 production by FLS (Figure 3B) were significantly inhibited in transfected FLS compared with control FLS. Moreover, the reduced proliferation of and IL-6 production by FLS could be rescued by Cyr61 protein (Figures 3A and B). This result suggested that miR-22 negatively regulates RA FLS proliferation and IL-6 production, and that this effect is mediated through suppression of Cyr61 expression. Furthermore, we used a specific anti–miR-22 to neutralize endogenous miR-22 in FLS and observed that anti–miR-22 efficiently enhanced FLS proliferation and production of IL-6 (Figures 3C and D).

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Figure 3. MicroRNA-22 negatively regulates fibroblast-like synoviocytes (FLS) proliferation and interleukin-6 (IL-6) production. A, Proliferation of carboxyfluorescein succinimidyl ester (CFSE)–labeled FLS transfected with negative control (left), miR-22 (middle), or miR-22 plus Cyr61 protein (right), as determined by flow cytometry. B, IL-6 production by FLS treated as in A, as determined by enzyme-linked immunosorbent assay (ELISA). C, Proliferation of CFSE-labeled FLS transfected with negative control (left) or anti–miR-22 (right), as determined by flow cytometry. D, IL-6 production by FLS treated as in C, as determined by ELISA. E, Phosphorylation of Akt in FLS transfected with negative control, miR-22, miR-22 plus Cyr61 protein, or anti–miR-22, as determined by Western blotting. Values in B and D are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for other definitions.

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Because Akt signaling is a main downstream effector of Cyr61 activation ([31]), we also examined Akt activation in the experiments described above. The results showed that phosphorylation of Akt was enhanced in the FLS transfected with anti–miR-22 (Figure 3E), while phosphorylation of Akt was inhibited in the FLS transfected with miR-22 but could be restored by Cyr61 protein (Figure 3E). Taken together, these data indicate that miR-22 negatively regulates FLS proliferation and IL-6 production by targeting Cyr61.

WT p53 binds to the promoter region of miR-22 and enhances miR-22 expression

A previous study showed that in the setting of tumor development, p53 signaling regulates miR-22 expression ([27]). Therefore, we sought to determine whether miR-22 expression in RA is also controlled by p53. We first examined the effect of WT p53 on the expression of miR-22 in RA FLS. The results showed that miR-22 expression in FLS transfected with WT p53 plasmid was increased compared with that of pEGFP-N3 control (Figure 4A). Moreover, we used a specific p53 siRNA (see Supplementary Figure 1, available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract) to knock down p53 expression in FLS and observed that miR-22 expression was reduced significantly in FLS with p53 knockdown (Figure 4B).

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Figure 4. Wild-type p53 enhances the transcriptional activity of miR-22 by binding to the promoter region of miR-22. A, Expression of miR-22 in fibroblast-like synoviocytes (FLS) transfected with pEGFP-N3 or WT p53, as detected by real-time polymerase chain reaction (PCR) using TaqMan probes. B, Expression of miR-22 in FLS transfected with small interfering RNA (siRNA) for negative control (SiNC) or p53 (Sip53), as detected by real-time PCR. C, Schematic representation of C17orf91 (miR-22 host gene) and putative p53 binding sites in the 5′ upstream region and intron 2 of C17orf91. D, Luciferase activity of different promoter constructs in human skin fibroblasts (HSFs) transfected with WT p53 or empty vector. Solid circles show show the position of the p53 binding site. E, Chromatin immunoprecipitation (ChIP) assay of p53 occupancy on the miR-22 promoter in HSFs transfected with WT p53 vector. Values in A, B, and D are the mean ± SD of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for other definitions.

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We next sought to determine whether p53 regulates transcription of the miR-22 gene by directly binding to its promoter region. According to bioinformatics analysis and a previous report ([27]), putative p53 binding sites are located in a 5′ upstream region and within intron 2 of C17orf91 (the miR-22 host gene) (Figure 4C). The sequences representing the 1.0K, 1.2K, and 1.3K regions in the 5′ region of C17orf91 and a 3.4K region containing both the 5′ and the intron 2 p53 sites were cloned into the luciferase reporter vector pGL3 to generate miR-22-1.0K, miR-22-1.2K, miR-22-1.3K, and miR-22-3.4K plasmids. The results of luciferase assays showed that cotransfecting WT p53 plasmid with the miR-22-1.2K, miR-22-1.3K, and miR-22-3.4K plasmids into Hs27 cells resulted in remarkable enhancement of luciferase activity compared with control (Figure 4D). Interestingly, the miR-22-3.4K construct exhibited stronger activation by WT p53, supporting the idea that both p53 binding sites (in the upstream region and in intron 2) are functional.

We also performed ChIP assays to detect the binding of p53 at endogenous miR-22 promoters in vivo. Four PCR amplicons were designed: 2 flanking the p53 site in the 5′ upstream region of C17orf91 (5′ #1 and 5′ #2), and 2 flanking the p53 site located in intron 2 of C17orf91 (Int #1 and Int #2). Indeed, p53 binding to the p53 binding sites located in the 5′ upstream region and intron 2 was significantly enhanced after transfection with WT p53, as shown in Figure 4E. Collectively, our results suggested that WT p53 controls miR-22 transcription by binding to the miR-22 gene locus.

Somatic mutation of p53 in RA leads to reduced miR-22 expression, contributing to Cyr61 overexpression

Given the presence of somatic mutations of p53 in RA synovial tissue, which are believed to be involved in synovial hyperplasia in RA ([8-13, 32, 33]), and our finding that p53 regulates miR-22 expression, we hypothesized that these p53 mutations may lead to dysregulated miR-22 expression, which could in turn contribute to the overexpression of Cyr61 in RA FLS. We first used exon sequencing to systematically analyze somatic mutations of p53 in exons 2–11 in 37 samples of RA synovial tissue. Our results showed that among the tested RA specimens, 73% harbored p53 mutations. For instance, 70.3% of these specimens contained the codon 72 Pro (CCC)–to–Arg (CGC) mutation in exon 3, and among these specimens, 2 contained the codon 47 Pro (CCG)–to–Arg (CGG) mutation in exon 3 simultaneously; 2.7% of the specimens contained the codon 367 Ser (AGC)–to–Asn (AAC) mutation in exon 10 (see Supplementary Table 3, available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract).

To further study the potential effect of these p53 mutations on miR-22 expression, we constructed plasmids expressing WT p53 or the 3 mutant forms of p53 detected in our exon sequencing (Figure 5A), in which the p53 coding sequence was fused to that of green fluorescent protein. As shown in Supplementary Figure 2 (available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract), all of the plasmids were successfully transfected. Compared with WT p53, the mutant forms of p53 (Figure 5A) failed to enhance the luciferase activity of miR-22-1.2K, miR-22-1.3K, and miR-22-3.4K reporters in Hs27 cells (Figure 5B). Consistently, real-time PCR analysis showed increased expression of miR-22 in FLS transfected with WT p53 but not the mutant forms of p53 (Figure 5C). Next, we examined the expression of Cyr61 in FLS transfected with the p53 plasmids and observed that transfection with WT p53 significantly decreased the expression of Cyr61 in FLS, whereas the mutant forms of p53 had little effect (Figure 5D). We therefore concluded that mutant forms of p53 in RA synovial tissue exhibit an impaired ability to promote miR-22 expression, and that the resulting reduction in miR-22 levels contributes to Cyr61 overexpression in RA.

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Figure 5. Expression of miR-22 is suppressed by mutant p53. A, Schematic representation of the 3 mutant plasmids constructed in p53 exons. Three mutant p53 plasmids (m1, m2, m3) were constructed based on the results of p53 exon sequencing, in which the p53 coding sequence was fused to that of green fluorescent protein. B, Luciferase activity of different miR-22 promoter constructs in human skin fibroblasts transfected with empty vector, WT p53, or the 3 mutant p53 vectors shown in A. C, Expression of miR-22 in fibroblast-like synoviocytes (FLS) transfected with empty vector, WT p53 vector, or the 3 mutant p53 vectors, as analyzed by real-time polymerase chain reaction using TaqMan probes. D, Western blot analysis of Cyr61 expression in FLS transfected with pEGFP-N3, WT p53 vector, or the 3 mutant p53 plasmids. Values in B and C are the mean ± SD of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01. See Figure 1 for other definitions.

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Mutant forms of p53 are functionally defective in RA synovial tissue

Finally, to further corroborate that mutant forms of p53 in RA synovial tissue represent loss-of-function mutations, we examined the expression of p53 target genes in RA synovium. The genes of both p21, a potent cyclin-dependent kinase inhibitor that functions as a regulator of cell cycle progression at G1 ([34]), and Bax, a protein of the Bcl-2 gene family that promotes apoptosis by competing with Bcl-2 ([35]), are regulated by p53. We thus investigated the expression of p21 and Bax. The results showed that the expression of p21 and Bax in the synovial tissue of RA patients was significantly lower than that in the synovial tissue of patients with OA, who do not harbor p53 mutations ([32]) (Figures 6A and B). In contrast, the expression of Cyr61 was remarkably increased in RA specimens. Significant negative correlations were observed between the expression levels of Cyr61 and p21 and the expression levels of Cyr61 and Bax (Figures 6C and D). These results were consistent with the notion that the p53 mutants in RA synovial tissue represent a loss of function.

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Figure 6. Mutant forms of p53 are functionally defective in rheumatoid arthritis (RA) synovial tissue. A and B, Expression of p21 (A) and Bax (B) in osteoarthritis (OA) and RA synovial tissue, as detected by real-time polymerase chain reaction. Values are the mean ± SD. ∗ = P < 0.05. C and D, Negative correlation between the expression of p21 and Cyr61 (P < 0.05) (C) and between Bax and Cyr61 (P < 0.01) (D). E, Expression of p53 in RA and OA synovial tissue, as determined by Western blotting. F, Schematic representation of wild-type (WT) and mutant p53 signaling in miR-22 and Cyr61 expression. FLS = fibroblast-like synoviocytes; IL-6 = interleukin-6.

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It is known that WT p53 is hardly detectable due to its very short half-life of <30 minutes, while mutant forms of p53 often display a prolonged half-life, thereby permitting their detection ([36]). Therefore, we performed Western blotting to examine the protein levels of p53 in RA and OA synovial tissue and observed that p53 was abundantly expressed in RA synovial tissue, whereas little or no immunoreactive p53 protein could be detected in OA synovial tissue (Figure 6E). This further supported the concept that the p53 molecule in RA synovium is likely to be mutated. The expression of p53 and Cyr61 in synovial tissue sections from patients with RA was also detected by immunofluorescence (see Supplementary Figure 3, available at the Arthritis & Rheumatology Web site at http://onlinelibrary.wiley.com/doi/10.1002/art.38142/abstract). As shown in Figure 6F, WT p53 can bind to the promoter region of miR-22 and induce miR-22 expression. MiR-22 may further bind to the 3′-UTR of Cyr61, leading to degradation of Cyr61. However, mutant p53 could not bind to the promoter region of miR-22 and repress miR-22 expression, leading to inhibition of Cyr61 degradation. The increased expression of Cyr61 promotes FLS proliferation and IL-6 production via the Akt signaling pathway. Taken together, our findings support the notion that the mutant forms of p53 observed in RA synovial tissue are functionally defective.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

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 ([4]). 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 ([5]). 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 ([21]), 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 ([27]). 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 ([46]). However, these p53 variants have been shown to influence the function of p53 in apoptosis, cell cycle control, and DNA repair ([47]). 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 ([50]), and that p53 mutations in the synovial tissue were associated with increased local expression of IL-6 ([13]). 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 ([5]).

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. 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.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ART_38142_sm_SupplTable1.doc39KSupplementary Table 1
ART_38142_sm_SupplTable2.doc62KSupplementary Table 2
ART_38142_sm_SupplTable3.doc82KSupplementary Table 3
ART_38142_sm_SupplFigure1.tif31KSupplementary Figure 1
ART_38142_sm_SupplFigure2.tif260KSupplementary Figure 2
ART_38142_sm_SupplFigure3.tif321KSupplementary Figure 3
ART_38142_sm_SupplFigure4.tif66KSupplementary Figure 4

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