Abstract
Objective. Interleukin 34 (IL-34) and microRNA 21 (miR-21) were found to be involved in the pathological process of rheumatoid arthritis (RA), but the details were unclear. In this study, we aimed to clarify the relationship between IL-34 and miR-21 in RA.
Methods. IL-34 concentrations in serum and synovial fluid (SF) of patients with RA were measured by ELISA. Fibroblast-like synovial cells (FLS) were cultured for evaluation of STAT3 activation, miR-21, and Bax/Bcl-2 expression by Western blot and real-time PCR. Correlations were analyzed between clinical features and detectable variables including SF IL-34 levels and miR-21 expression.
Results. SF IL-34 levels were significantly higher in patients with RA who had a high 28-joint Disease Activity Score (DAS28 ≥ 3.2) than in those with a lower DAS28 (DAS28 < 3.2). DAS28 scores and miR-21 expression in FLS had a significant positive correlation with the SF IL-34 levels. In addition, IL-34 stimulation strengthened the activation of p-STAT3, resulting in the increment of miR-21 expression. Inhibiting of miR-21 expression contributed to decreased Bcl-2/Bax ratio, suggesting that miR-21 was involved in the resistance to apoptosis. With the blocking of the colony-stimulating factor-1 receptor (CSF1R), decreased protein expressions including CSF1R, p-STAT3/STAT3, and Bcl-2/Bax were shown, suggesting that CSF1R participated in the biological functions of IL-34 in RA.
Conclusion. The IL-34/STAT3/miR-21 pathway is crucial for the survival of synovial fibroblasts in RA, which might be candidate therapeutic targets for RA treatment.
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized mainly by chronic synovial inflammation1. Synovial fibroblasts, the major cell population in synovial tissue, are involved and actively participate in the pathogenesis of RA2. In more recent years, interleukin 34 (IL-34), as a newly discovered cytokine, has shown that it can be locally expressed in the synovial tissue and is associated with synovitis severity of RA3. The positive correlation between IL-34 levels and rheumatoid factor (RF) and anticyclic citrullinated peptide antibody titers was also indicated4,5. However, for IL-34, the precise mechanism by which it carries out its biological functions in RA is still unknown.
MicroRNA (miRNA), small noncoding RNA molecules, serve as key mediators of gene silencing at the posttranscription level in fundamental biological and pathological processes6. A report showed that miRNA 21 (miR-21) was involved in the imbalance of Th17/Treg cells by regulating STAT3/STAT5 in patients with RA7. In addition, high expressions of Bcl-2 and miR-21 in synovial fluid (SF) Treg cells of patients with RA were found, suggesting that miR-21 might participate in the apoptotic progress and regulation of RA pathogenesis8. Regrettably, few details are known about the clinical significance of miR-21 in RA, and the expression and function of miR-21 in fibroblast-like synovial cells (FLS) also remain unclear.
In our study, we found that stimulation with IL-34, binding to colony-stimulating factor-1 receptor (CSF1R), induced increment of miR-21 expression through p-STAT3 activation, which was associated with disease activity of RA. Inhibiting of miR-21 resulted in increased apoptosis in the FLS, suggesting that miR-21 was involved in the pathogenesis of RA. Our findings suggested that the IL-34/STAT3/miR-21 pathway was crucial for the survival of synovial fibroblasts, which might be candidate therapeutic targets for the treatment of RA.
MATERIALS AND METHODS
Patients
Thirty patients with RA were enrolled from the Department of Integrated Chinese Traditional and Western Medicine at the Tongji Hospital, affiliated with the Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China)9. Twenty patients with osteoarthritis (OA) and 20 age- and sex-matched healthy individuals served as controls. Our study was approved by the Ethics Committee of the Tongji Medical College according to the Declaration of Helsinki and written informed consent was given by all patients.
The 4 variables used to calculate the 28-joint Disease Activity Score (DAS28) included the number of swollen joints, number of tender joints, C-reactive protein or erythrocyte sedimentation rate, and the patient’s global assessment. A DAS28 score of 3.2 and above indicated moderate to high disease activity whereas a value of below 3.2 referred to remission to low disease activity10.
Isolation and culture of FLS
FLS were isolated by enzymatic digestion of synovial tissue from patients with RA and OA undergoing total joint replacement surgery. Briefly, synovial tissue was minced and incubated for 2 h at 37°C with 1 mg/ml collagenase (Sigma-Aldrich Co.) in Dulbecco modified Eagle’s medium (Hyclone) containing 10% fetal bovine serum (Hyclone). The cell suspension was filtered through a 70-μm nylon cell strainer (BD Falcon). Cells were sequentially subcultured for 3–6 passages and used as the source of FLS.
Different concentrations of IL-34 (0, 10, 20, and 50 ng/ml; R&D Systems) were used at indicated times as stimulators of FLS. Unless specifically mentioned, STA21 (STAT3 inhibitor, 50 μM; Santa Cruz Biotechnology) and human CSF1R-blocking antibody (20 ng/ml; Novus Biologicals) were used in our study.
ELISA
IL-34 levels in serum and SF samples were measured by ELISA according to the manufacturer’s directions (R&D Systems).
Flow cytometry detection of apoptosis
The apoptotic cells were detected using an FITC Apoptosis Detection Kit (BD Pharmingen). FLS were collected, washed, and resuspended in buffer containing 5 µL of FITC annexin V and 5 µL of propidium iodide (PI). Following incubation of 15 min in the dark at room temperature, fluorescence of the cells was analyzed by BD FACSCanto II using DiVa software (BD Bioscience). Apoptosis cells contained the early apoptotic cells (annexin V-positive and PI-negative) and the late apoptotic or necrotic cells (both annexin V-positive and PI-positive).
RNA isolation and real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed using M-MLV-RTase (Promega) according to the manufacturer’s instructions. Real-time PCR was performed using the SYBR-Green Master PCR Mix (Applied Biosystems) on the TP800 qPCR System (Takara). Real-time PCR of cDNA was performed using the forward (F) and reverse (R) primer sequences: miR-21: F, 5′-TGT CGG GTA GCT TAT CAG ACT-3′; R, 5′-TGT CAG ACA GCC CAT CGA CT-3′; U6: F, 5′-CTC GCT TCG GCA GCA CA-3′; R, 5′-AAC GCT TCA CGA ATT TGC GT-3′. The expression of U6 was used as endogenous control for miRNA analysis. β-actin: F, 5′-TCA TGA GGT AGT CAG TCA GG-3′; R, 5′-CTT CTA CAA TGA GCT GCG TG-3′; Bcl-2: F, 5′-TCC GAT CAG GAA GGC TAG AGT T-3′; R, 5′-TCG GTC TCC TAA AA GCA GGC-3′; Bax: F, 5′-CCG CCG TGG ACA CAG AC-3′; R, 5′-CAG AAA ACA TGT CAG CTG CCA-3′. The relative Bax or Bcl-2 mRNA levels were normalized to those of β-actin for mRNA analysis. For relative quantification, 2−(Ct-Cc) was used to calculate the relative expression (Ct and Cc are the mean threshold cycle differences after normalizing to control that was calculated and used as an indication of the relative expression levels11).
Western blot analysis
FLS from patients with RA was collected and washed 3 times with sterile phosphate buffered saline, and then lysed with 0.1–0.2 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% NaN3, 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM sodium orthovanadate, and 1 mM NaF). Equal amounts of proteins were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) presoaked with 100% methanol. This was followed by incubation with mouse anti-human CSF1R monoclonal antibody (Novus Biologicals) and the rabbit anti-human antibody against Bcl-2, Bax, STAT3, p-STAT3 (Tyr705), and β-actin (Cell Signaling Technology Inc). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin G secondary antibodies were used, respectively. Finally, immunoblot signals were visualized using a scanner (HP Scanjet 7400C, Hewlett-Packard), and then imaged and quantitated using a ChemiScope 3400 Mini (CLINX Science Instruments).
Transfection
Transfection was performed using the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. MiR-21 inhibitor (5’-UCA ACA UCA GUC UGA UAA GCU A-3’) and negative control (5’-CAG UAC UUU UGU GUA GUA CAA-3’) were synthesized by Genepharma. All of the oligonucleotides were transfected at a final concentration of 50 mM.
Statistical analysis
All data are presented as mean ± SD. Paired Student t test was used to compare variables between groups. Pearson correlation coefficient was used to test the correlations analysis. P < 0.05 was considered statistically significant.
RESULTS
SF IL-34 levels and its association with miR-21 expression in FLS from patients with RA with moderate to high disease activity
First, we aimed to learn the relationship of SF IL-34 levels and miR-21 expression in FLS. Clinical characteristics of patients with RA are shown in Table 1. As shown in Figure 1A, serum IL-34 levels were much higher among the patients with RA (n = 30) compared with the healthy controls (n = 20, p < 0.001). SF IL-34 levels were markedly higher in patients with RA (n = 30) than in patients with OA (n = 20, p < 0.001; Figure 1B). Patients with RA with moderate to high disease activity (DAS28 ≥ 3.2, n = 17) had significantly higher mean SF IL-34 levels than did those with low disease activity (DAS28 < 3.2, n = 13, p < 0.05; Figure 1C). The miR-21 expression of FLS samples from patients with RA with moderate to high disease activity (DAS28 ≥ 3.2, n = 12) was elevated compared with patients with OA (n = 6) and patients with RA with low disease activity (DAS28 < 3.2, n = 6, p < 0.05; Figure 1D).
On linear regression analysis, SF IL-34 levels had a significant positive relationship with miR-21 expression (r = 0.909, p < 0.01; Figure 1E). Additionally, miR-21 expression was significantly associated with disease activity in patients with RA (r = 0.95, p < 0.01; Figure 1F). The correlation between the clinical features and detectable variables including SF IL-34 and miR-21 expression are also shown in Table 2.
STAT3 activation contributes to IL-34–dependent miR-21 expression
Based on the correlation analysis, we wanted to indicate which 1 gene activation was involved in the continuous IL-34 stimulation. As shown in Figure 2A, treatment with IL-34 significantly decreased apoptosis of FLS by flow cytometric analysis. A representative Western blotting showed that expression of p-STAT3 was evidently increased in a dose-dependent manner upon IL-34, and miR-21 expression in FLS was also raised in response to stimulation of IL-34 (Figure 2B, Figure 2C). Of note, treatment with STA21, a STAT3 inhibitor, abrogated IL-34–induced p-STAT3 activation and miR-21 expression, suggesting that STAT3 activation contributed to IL-34–dependent miR-21 expression (Figure 2D).
MiR-21 is involved in the resistance to apoptosis of IL-34
Considering the involvement of miR-21 in apoptosis, we continued to examine its effect on apoptosis-associated genes in synovial fibroblasts. As shown in Figure 3A, expression of antiapoptotic Bcl-2 was higher in FLS from patients with RA than patients with OA, and expression of proapoptotic Bax was lower (Figure 3B). The similar tendency was discovered in the patients with RA with different disease activity (Figure 3A, Figure 3B). Following the stimulation of IL-34, the expression of Bcl-2 increased in a dose-dependent manner, but the expression of Bax was decreased (Figure 3C, Figure 3D). Interestingly, transfection of miR-21 inhibitor resulted in decrement of the Bcl-2/Bax ratio, suggesting that miR-21 was involved in the regulation of Bcl-2/Bax (Figure 3E).
CSF1R participates in the biological activities of IL-34 in RA
We aimed to clarify whether CSF1R was involved in the biological functions of IL-34. With IL-34 stimulation, increased CSF1R expression in the FLS was found (Figure 4A). Simultaneously, miR-21 expression was enhanced in the IL-34–dependent manner, which was noticeably limited in the presence of CSF1R-blocking antibody (Figure 4B, Figure 4C). Importantly, the declining protein expressions including CSF1R, p-STAT3/STAT3, and Bcl-2/Bax were displayed with the treatment of CSF1R-blocking antibody, suggesting that CSF1R participated in the biological function of IL-34 in RA (Figure 4D).
DISCUSSION
In our study, we demonstrate that upregulated IL-34 can strengthen STAT3 activation, resulting in increments of miR-21 expression by binding to CSF1R, which regulate the change of Bcl-2/Bax ratio and contribute to the resistance to apoptosis of FLS in RA.
IL-34, produced by synovial fibroblasts in the rheumatoid joints, might be involved in the pathogenesis of RA12. Our results showed that serum and SF IL-34 levels were increased significantly in patients with RA, as a previous paper described (Figure 1A, Figure 1B)4. In our study, patients with RA were divided into 2 groups according to the DAS28 scores: moderate to high disease activity (DAS28 ≥ 3.2) and low disease activity (DAS28 < 3.2). Patients with RA with a higher DAS28 (DAS28 ≥ 3.2) had significantly higher mean SF IL-34 levels and miR-21 expression than those with a lower DAS28 (DAS28 < 3.2; Figure 1C, Figure 1D). Additionally, miR-21 expression had a significant positive relationship with the SF IL-34 levels in patients with RA with moderate to high disease activity (Figure 1E). To our knowledge, ours is the first study to show the difference of SF IL-34 levels based on the DAS28 scores in patients with RA. The positive relationship of DAS28 scores and SF IL-34 levels suggest that elevated SF IL-34 might be a useful marker to evaluate active RA (Table 2).
The relation between miR-21 and STAT3 has been described in a paper13. In RA synovial tissue, STAT3 increased in proportion to the severity of synovitis14. In Sézary syndrome, miR-21 expression in CD4+ T cells was regulated by STAT3, and the silencing of miR-21 resulted in increased apoptosis15. In our current study, treatment with IL-34 significantly decreased apoptosis of FLS by flow cytometric analysis (Figure 2A). However, IL-34 stimulation enhanced the activation of STAT3 in a dose-dependent manner (Figure 2B). Consistent with the result, the miR-21 expression was increased upon IL-34, but was hardened in the presence of STAT3 inhibitor (Figure 2C, Figure 2D). These findings suggested that IL-34 contributed to the increment of miR-21 expression through STAT3 activation. On the contrary, Dong, et al reported that the decreased expression of miR-21 was correlated with the imbalance of Th17 and Treg cells in patients with RA7. Although accompanied by STAT3 activation, the levels of miR-21 expression were significantly lower in peripheral blood mononuclear cells (PBMC) and CD4+ T cells of patients with RA. Compared with Dong, et al7, there were some differences in our study. First, we enrolled patients with RA who had moderate to high disease activity. The individual differences including duration time of RA, drugs for treatment such as methotrexate, and baseline health status might affect the results. Second, FLS were separated and cultured as detected samples, but not PBMC and CD4+ T cells. Additionally, there was no positive correlation between SF IL-34 and miR-21 expression in CD4+ T cells (or PBMC) from patients with RA in our study (data not shown). Third, FLS were subsequently stimulated with different concentration of IL-34, unlike lipopolysaccharide used by Dong, et al.
T cells from synovial tissue of patients with RA were resistant to apoptosis because of the high expression of Bcl-216. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21, which induced little apoptosis in comparison with SF-conventional T cells and peripheral blood Treg cells17. Our results also showed that the proapoptotic Bax was decreased and antiapoptotic Bcl-2 was increased, but the change can be reversed by transfection with miR-21 inhibitor (Figure 3). We speculate that IL-34 stimulation induced the STAT3 activation, and subsequently increased miR-21 expression, leading to an upregulated Bcl-2/Bax ratio, which contributed to limited apoptosis in FLS of RA. Further study needs to be designed to explain the details, which would help to clarify the significance and function of IL-34 and miR-21 in RA.
IL-34 was identified as an alternative CSF1R ligand and shared functional similarities with macrophage colony stimulating factor (M-CSF)18. It was discovered that CSF1R expression was increased in the synovium of patients with RA, and the blocking of CSF1R protected against bone and cartilage destruction in the collagen-induced arthritis model19. In our paper, IL-34 stimulation mediated p-STAT3 activation by binding to CSF1R and induced the increased expression of miR-21, which contributed to the upregulated Bcl-2 and downregulated Bax expressions. Inhibition of miR-21 restricted the IL-34/STAT3/miR-21 pathway and effectively promoted the expression of Bax, resulting in increased apoptosis. Compared with the miR-21 inhibitor, treatment with CSF1R-blocking antibody restrained the expression of miR-21 and Bax, suggesting that CSF1R participated in the IL-34/STAT3/miR-21 pathway (Figure 4). Other researchers reported that protein tyrosine phosphatase-ζ (PTP-ζ), a novel IL-34 receptor, increased the biological effects of IL-3420. In addition, IL-34 could induce the migration of syndecan-1–expressing cells, indicating syndecan-1 as a new molecular actor in the M-CSF/IL-34/M-CSFR triad21. More studies are required for further clarification of whether other IL-34 receptors, PTP-ζ and syndecan-1, are involved in the binding activity of IL-34 or present a cross-reaction to affect miR-21 expression.
Our results showed that IL-34 could induce STAT3 activation by binding to CSF1R and contributing to the increment of miR-21 expression, which was associated with disease activity of RA. Further, increased miR-21 expression resulted in an improved Bcl-2/Bax ratio, which was involved in limited apoptosis of FLS. These findings suggest that the IL-34/STAT3/miR-21 pathway is crucial for the survival of synovial cells, which might be alternative targets for the treatment of RA.
Footnotes
Supported by grants from the National Natural Science Foundation of China (No. 81503426).
- Accepted for publication March 1, 2016.