Nesfatin-1 facilitates IL-1β production in osteoarthritis synovial fibroblasts by suppressing miR-204-5p synthesis through the AP-1 and NF-κB pathways

The progression of osteoarthritis (OA) is mediated by adipokines, one of which is nesfatin-1, which is responsible for the production of inflammatory cytokines. However, how this molecule may affect the synthesis of the proinflammatory cytokine interleukin 1 beta (IL-1β) in OA is unclear. Our analyses of records from the Gene Expression Omnibus (GEO) dataset and clinical specimens of synovial tissue revealed higher levels of nesfatin-1 and IL-1β in OA samples compared with normal healthy tissue. We found that nesfatin-1 facilitates IL-1β synthesis in human OA synovial fibroblasts (OASFs) and suppresses the generation of micro-RNA (miR)-204-5p, as the miR-204-5p levels in OA patients were lower than those in healthy controls. Nesfatin-1-induced stimulation of IL-1β in human OASFs occurred via the suppression of miR-204-5p synthesis by the PI3K, Akt, AP-1 and NF-κB pathways. We suggest that nesfatin-1 is worth targeting in OA treatment.


INTRODUCTION
The main feature of osteoarthritis (OA) is the diseaserelated increase in inflammatory mediators in the synovial membrane leading to pannus formation and resulting in cartilage degradation and bone destruction [1,2]. The generation of proinflammatory cytokines and chondrolytic enzymes in the inflamed synovial tissue and cartilage degradation promotes inflammation in the synovium [3][4][5]. OA synovial fibroblasts (OASFs) enhance arthritic pathology by increasing levels of chondrolytic enzymes and inflammatory cytokines [6,7].
AGING that cause ongoing damage in the synovium, bone and cartilage [10,11]. Among these inflammatory mediators, interleukin 1 beta (IL-1β) contributes to the pathogenesis of OA by facilitating proteolytic enzymeinduced damage in the cartilage extracellular matrix [2]. Levels of IL-1β in OA synovial fluid and serum are higher than those in normal healthy individuals and have therefore been targeted by therapies such as the IL-1β inhibitor canakinumab [12]. Reducing the activity of proinflammatory cytokines has been shown to slow the progression of arthritis [2], as these cytokines and also matrix-and cartilage-degrading enzymes account for pathohistological changes that occur in OA [13][14][15]. These inflammatory mediators are regulated by different signaling pathways in OA, such as the phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), activator protein 1 (AP-1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) pathways [16]. In particular, the PI3K/Akt/mTOR signaling pathway is essential for maintaining joint health, and correlates with the degradation of cartilage in OA pathogenesis, as this pathway is involved in chondrocyte apoptosis, proliferation and cytokine production [16]. The phosphorylation of PI3K/Akt signaling can promote the translocation of AP-1 and NF-B into the nucleus and increases gene expression of proinflammatory mediators, such as prostaglandin E2 and cyclooxygenase-2 [17][18][19]. Thus, examining the PI3K/Akt, AP-1 and NF-B pathways is expected to improve our understanding as to how to reduce inflammatory cytokine expression in OA.
Adipose tissue is an endocrine organ that produces hormones as adipokines including adiponectin, leptin, resistin and nesfatin-1 [28]. Nesfatin-1 plays a crucial role in food intake and weight control [29] and regulates different cellular functions such as growth, migration, differentiation and apoptosis in mammalian cells [30]. Nesfatin-1 levels in serum and synovial fluid correlate with the severity of knee OA [31]. Nesfatin-1 also suppresses the destruction of cartilage and ameliorates OA in rats [32]. However, the effects of nesfatin-1 on IL-1β expression in OASFs is unknown. In this study, our comparison of nesfatin-1 and IL-1β levels found that both were higher in OA patients than in healthy normal controls, and our investigations revealed that nesfatin-1 promotes IL-1β synthesis in OASFs by suppressing miR-204-5p expression in the PI3K, Akt, AP-1 and NF-κB pathways. Targeting nesfatin-1 levels in synovial fibroblasts may assist in the management of OA disease.

Higher levels of nesfatin-1 and IL-1β expression in OA patients
Concentrations of nesfatin-1 in human serum and synovial fluid reflect the severity of OA [31]. Gene Expression Omnibus (GEO) database records revealed higher levels of nesfatin-1 and IL-1β in synovial tissues from OA patients compared with samples from healthy controls ( Figure 1A and 1B). Our clinical samples also confirmed higher levels of nesfatin-1 and IL-1β in serum from OA patients compared with serum from healthy individuals ( Figure 1C and 1D), indicating that nesfatin-1 and IL-1β are associated with progression of OA.

DISCUSSION
OA is well recognized for its characteristics of joint degradation and synovial membrane inflammation [34]. OASFs in the joint microenvironment are critical to the progression of OA, increasing proinflammatory cytokine production, which leads to cartilage degradation and bone erosion [34]. Numerous commercially available arthritis therapeutics target inflammatory cytokines, including IL-1β [35]. Adipokines are produced and secreted by adipose tissue and are involved in many physiological activities, including glucose and lipid metabolism, and also immune and inflammatory responses [36]. In OA, adipokines (including leptin, adiponectin and resistin) AGING are considered to be useful therapeutic targets, as they can regulate proinflammatory cytokine expression and thus influence OA development [37][38][39][40]. A recent study reported that adiponectin is capable of inducing gene expression of monocyte chemoattractant protein-1 (MCP-1), IL-6 and matrix metalloproteinase 1 (MMP-1) in human OA chondrocytes [38], while leptin increases levels of IL-1, IL-8 and MMPs in chondrocytes as well as IL-6 and MMP-13 expression in synoviocytes, so contributes to cartilage catabolism in OA [41]. Resistin can activate the p38-MAPK, NF-κB and cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathways and thus stimulate proinflammatory cytokine and chemokine expression in OA disease [42]. Nesfatin-1 has demonstrated anti-inflammatory activities [32], although the effect of nesfatin-1 on IL-1β production in OA is uncertain. Our study demonstrates that human OA synovial tissue contains higher levels of nesfatin-1 and IL-1β compared with tissue from healthy individuals. Cellular investigations revealed that nesfatin-1 promotes IL-1β synthesis. We also confirmed that nesfatin-1 increases IL-1β concentrations in human OASFs by suppressing IL-1β synthesis in the PI3K/Akt, AP-1 and NF-κB pathways.
PI3K/Akt activation is crucial in the adjustment of numerous cellular roles [43]. Notably, the PI3K/Akt signaling cascade mediates IL-1β expression during the progression of arthritis disease [25,44,45]. Our data show that nesfatin-1 promotes the phosphorylation of PI3K and Akt, while their respective inhibitors inhibit nesfatin-1-induced IL-1β production in human OASFs. This was confirmed by similar results with p85 and Akt siRNAs. Our evidence reveals that activation of PI3K/Akt signaling controls nesfatin-1-enhanced promotion of IL-1β synthesis during OA development.
Numerous transcription factor binding elements have been reported in the promoter site of IL-1β [46,47]. In particular, AP-1 and NF-κB mediate IL-1β transcription and inflammatory responses [25,46]. We found that inhibitors of AP-1 (tanshinone IIA) and NF-κB (PDTC and TPCK) suppressed nesfatin-1-induced production of IL-1β in human OASFs. Confirmation of these effects by genetic inhibition using c-Jun and p65 siRNAs indicated that AP-1 and NF-κB transcriptional activation is mediated by nesfatin-1-induced synthesis of IL-1β. We also observed that nesfatin-1 enhances c-Jun and p65 phosphorylation, as well as AP-1 and NF-κB luciferase activities. Pharmacological inhibitors of PI3K and Akt antagonized nesfatin-1-mediated activities, suggesting that nesfatin-1 promotes AP-1 and NF-κB-dependent IL-1β production and inflammatory responses through PI3K/Akt signaling. AGING miRNAs post-transcriptionally control gene synthesis [48]. During the development of arthritis disease, aberrant miRNA generation regulates inflammatory cytokine production and cartilage catalysis [48][49][50]. In particular, the mediation of IL-1β synthesis by miRNAs facilitates the progression of OA [25]. Our analysis of open-source databases identified 19 miRNAs that interfere with IL-1β transcription. Nesfatin-1 markedly inhibited levels of miR-204-5p expression. The analysis of records from the GEO database also found lower miR-204-5p levels in OA patients than in healthy controls. We enhanced miR-204-5p synthesis in human OASFs by transfecting them with a specific miR-204-5p mimic, which markedly inhibited IL-1β expression and cellular inflammatory responses. miR-204-5p expression was negatively associated with levels of IL-1β and proinflammatory cytokines in OA. Moreover, we found that inhibitors of PI3K (Ly2940002), Akt (Akti), AP-1 (tanshinone IIA) and NF-κB (PDTC and TPCK) can increase levels of miR-204-5p expression in human OASFs treated with nesfatin-1. Thus, the results indicated that miR-204-5p synthesis can be regulated by the PI3K/Akt pathway by facilitating the transcription of AP-1 and NF-κB into the nucleus of nesfatin-1treated human OASFs. Thus, our evidence has identified that miR-204-5p exhibits novel antiinflammatory properties, although the mechanisms of OA disease are complex and we only examined the in vitro effects of nesfatin-1 in human OASFs. Future work is needed to ascertain the in vivo effects of nesfatin-1 in OA. We suggest that further research should screen for a drug that can inhibit nesfatin-1 expression in inflammatory diseases such as OA.
In conclusion, our study has identified that nesfatin-1 facilitates IL-1β production in human OASFs by suppressing miR-204-5p synthesis in the PI3K/Akt, AP-1 and NF-κB pathways ( Figure 6). We believe that targeting nesfatin-1 may assist with the management of OA disease.

Bioinformatics analysis
Gene level profiles in OA and normal synovial tissue were obtained from the GEO database and analyzed for levels of nesfatin-1 and IL-1β expression.

Collection of clinical samples
Study approval by the Institutional Review Board (IRB) of China Medical University Hospital and all participants provided written informed consent before study enrolment (Approval Number: MUH108-REC3-039). Clinical samples were taken from 8 patients undergoing total knee arthroplasty for OA and from 8 patients undergoing arthroscopy after trauma/joint derangement (who served as healthy controls).

Western blotting analysis
SDS-PAGE resolved the total proteins, which were then transferred to the PVDF membranes, as per our previous publications [52][53][54][55]. The PVDF membranes were blocked with 4% non-fat milk in PBST for 1 h before incubating them with primary antibodies for 1 h, followed lastly by 1 h of incubation with HRPconjugated secondary antibodies. Finally, we examined the immunoblot band using an imaging system (ImageQuant ™ LAS 4000).

q-PCR analysis
Total RNA was extracted from OASFs with TRIzol agent and then transformed into complementary DNA using a reverse transcription kit. We conducted the qPCR analysis using PCR Master Mix. We used the SYBR ® RT-PCR kit and Mir-X ™ miRNA First-Strand Synthesis for reverse transcription of miRNAs. Analysis followed previous protocols [56][57][58].

ELISA assay
OASFs were treated with pharmacological inhibitors or transfected with siRNAs before being treated with nesfatin-1 for 24 h. IL-1β levels in the medium were then examined using the IL-1β ELISA kit, according to the manufacturer's procedures.

Statistical analysis
Values are shown as the mean ± standard deviation (S.D.). Significant differences between each group were assessed by the Student's t-test. All p values <0.05 were considered to be significant.