Follistatin‐like protein 1 (FSTL1) promotes chondrocyte expression of matrix metalloproteinase and inflammatory factors via the NF‐κB pathway

Abstract Background The expression of follistatin‐like protein 1 (FSTL1) is closely associated with diseases of the musculoskeletal system. However, despite being a well characterized inflammatory mediator, the effects of FSTL1 on chondrocytes are not completely understood. In this study, we investigated the effects of FSTL1 on the expression of inflammatory and catabolic factors in rat chondrocytes. Methods Rat chondrocytes were treated directly with various concentrations of FSTL1 in vitro. The levels of matrix metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)‐2, interleukin (IL)‐1β, tumour necrosis factor (TNF)‐α and IL‐6 were measured by polymerase chain reaction, ELISA and Western blotting. In addition, activation of the nuclear factor kappa B (NF‐κB) pathway was explored to identify potential regulatory mechanisms. Results Follistatin‐like protein 1 directly increased the expression of MMP‐1, MMP‐13, iNOS, COX‐2, IL‐1β, TNF‐α and IL‐6 at both gene and protein level in a dose‐dependent manner. Activation of NF‐ κB and phosphorylation of p65 were also promoted by FSTL1 stimulation. Conclusions Follistatin‐like protein 1 exerts pro‐inflammatory and catabolic effects on cultured chondrocytes via activation of the NF‐κB signalling pathway. FSTL1 may therefore be a target in the treatment of OA.

medications, the need for a structure-modifying treatment for OA remains.
Catabolic and inflammatory mediators are associated with the development of OA and have been targeted in drug studies. Diacerein, an inhibitor of IL-1β in vitro, was shown to be an effective treatment for patients with symptoms of OA. 4 Tanezumab, a newly developed monoclonal antibody against nerve growth factor, relieves pain and stiffness in patients with moderate-to-severe OA of the knee. 5 Thus, there is increasing interest in new therapies targeting OA.
Follistatin-like protein 1 (FSTL1), a secreted glycoprotein, was first identified by Shibanuma et al in 1993, as a transforming growth factor (TGF)-β-inducible protein. 6 Previous studies indicated that FSTL1 is involved in the progression of several diseases, including pulmonary fibrosis, cardiovascular disease, cancer and arthritis. [7][8][9][10][11] In the past decade, the importance of FSTL1 in the pathogenesis of arthritis has been determined, beginning with a study showing that anti-FRP antibody levels are higher in the synovial fluid and serum of rheumatoid arthritis (RA) patients than in patients with other systemic rheumatoid diseases. 12 The same group of researchers then reported that transformation of the TGF-β gene into synovial cells enhanced FSTL1 gene expression. And that FSTL1 protects against the joint erosion seen in RA by down-regulating the production of matrix metalloproteinase (MMP)-1, MMP-3 and prostaglandin E2. 13 Yury et al found a significant positive correlation between serum FSTL-1 levels and the arthritis index in mice with collagen-induced arthritis. The authors also reported that FSTL1 directly increases the expression of pro-inflammatory cytokines, such as interleukin (IL)-6 and MCP-1. 10

| Cells culture and treatment
A total of 18 rats (male:female, 1:1) were housed at 25˚C with 45%-75% relative humidity and enough food and water (12 hours light/dark cycle). After killed by intraperitoneal injection with 10% chloral hydrate (4 mL/kg; Sigma Aldrich; Merck KGaA, Darmstadt, Germany), the articular cartilage was removed from the tibial plateau and femoral condyle and minced into 1-3 mm 3 pieces. After digestion of the tissue using pronase and collagenase, the resulting cells were isolated by centrifugation and cultured as a monolayer in complete fresh Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% (v/v) foetal calf serum, 50 μg streptomycin/mL and 50 units penicillin/mL. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 and the medium was replaced every 2 days after seeding. All experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University, Zhejiang Sheng, China.
Chondrocytes at passage 2 (5 × 10 3 cells/well) were seeded in 96-well plates and incubated with various concentrations of FSTL1 (Sigma Aldrich; Merck KGaA) for 24 hours. Then, 20 μL of MTT (5 mg/mL) was added to each well. After a 4-h incubation, the culture medium was removed, an equal volume of DMSO was added to resuspend the formazan, and the absorbance at 570 nm was measured.

| Quantitative real-time polymerase chain reaction (RT-PCR)
MMP-1, MMP-3, MMP-13, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and tumour necrosis factor (TNF)-α expression was analysed using an RT-PCR system (Bio-Rad, Marnesla-Coquette, France). TRIzol reagent (Thermo Fisher Scientific, Inc, Waltham, MA, USA) was used according to the manufacturer's instructions to extract the total RNA of chondrocytes incubated with different concentrations of FSTL1 for 24 hours in 6-well plates. For the investigated genes, mRNA expression was normalized to that of GAPDH. The primers, amplicon length and annealing temperatures are described in Table 1. Relative gene expression was calculated using the 2 -∆∆Ct method.

| Enzyme-linked immunosorbent assay (ELISA)
Commercially available ELISA kits were used to quantify the levels of IL-1β, IL-6 and TNF-α in the medium (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's guidelines.

| Western blotting
The chondrocytes were washed with cold phosphate-buffered saline and lysed using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Cell proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and then transferred onto membranes. After the membranes had been blocked with 5% bovine serum albumin for 1 hour at room temperature, the proteins were probed overnight at 4°C using pri-

| Statistical analysis
All experiments were performed in triplicate using independent samples. The results are expressed as means ± SD and were analysed by one-way analysis of variance. Comparison between the groups was performed with post hoc Tukey's test. Statistical analyses were performed using SPSS for Windows software (version 19.0; IBM Corp., Armonk, NY, USA). A P < 0.05 was considered to indicate statistical significance.

| Effects of FSTL1 on cells viability
As shown in Figure 1, FSTL1 at a concentration of 0-25 ng/mL was not cytotoxic for the cultured chondrocytes. At FSTL1 concentrations of 50 and 100 ng/mL, cell viability decreased remarkably compared to non-treated cells. Therefore, in this study, FSTL1 was used at a dose of 0-25 ng/mL.

A significant up-regulation of these pro-inflammatory mediators in
cells incubated with 5 and 25 ng FSTL1/mL, at both gene and protein levels, was observed, especially in the high-dose FSTL1 group.
In cells treated with 1 ng FSTL1/mL, only the mRNA levels of IL-1β, TNF-α and IL-6 were increased.

| Effects of FSTL1 on NF-κB activation in chondrocytes
The potential mechanism underlying the observed effects of FSTL1 was explored by examining the effect of FSTL1 on the NF-κB signalling pathway ( Figure 5). Western blotting showed that, compared to the control group, the expression of p-NF-κB p65 and phosphorylation of IκBα were significantly enhanced in the FSTL1treated group.

| D ISCUSS I ON
Follistatin-like protein 1 (FSTL1) is a secreted extracellular glycoprotein involved in musculoskeletal system functions. In patients with intervertebral disc degeneration, serum FSTL1 levels were significantly higher than in the control group. In nucleus pulposus cells, FSTL1 induces the expression of the inflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and MMP-13, by activating both MAPK and NF-κB pathways. 16  Osteoarthritis is a chronic disease of the whole joint characterized by inflammation involving the cartilage, synovium, joint capsule, subchondral bone and meniscus. 19 IL-1β and TNF-α promote the production of MMPs and are thus key inflammatory mediators in OA progression. 20 However, both cytokines also inhibit the synthesis of extracellular matrix proteins. 21  The transcription factor NF-κB regulates the expression of a large number of inflammatory genes, as well as MMP production.
In the cytosol, NF-κB protein remains in an inactive state as long as it is bound to its inhibitor, IκBα. 34 In response to chemical signals or mechanical stress, the IκBα unit is phosphorylated and degraded, resulting in the nuclear translocation of NF-κB and the induction of gene transcription. 35 Therefore, NF-κB expression in chondrocytes may be a valuable target in the prevention and treatment of OA. 36 Previous studies showed that FSTL1 stimulates NF-κB signalling. 37,38 F I G U R E 3 Effects of FSTL1 on MMP-1, MMP-3, MMP-13, iNOS and COX-2 protein expression. Each column represents the mean ± SD. FSTL1 (1, 5 and 25 ng/mL) increased MMP-1, MMP-13 iNOS and COX-2 protein expression (P < 0.05), consistent with the RT-PCR results. A low dose of FSTL1 (1 ng/mL) had no effect on MMP-3 protein levels. *P < 0.05 vs control group The results of our Western blot analysis indicated that FSTL1 induces the activation of NF-κB p65 by promoting IκB-α degradation.
These findings suggest that the pro-inflammatory effects of FSTL1 in chondrocytes are mediated by its activation of NF-κB signalling.
A limitation of our study is that only in vitro model, monolayer cultured chondrocytes, is applied to mimic the development process of OA and evaluate the impact of pro-inflammatory factor FSTL1 on OA. Further research, such as 3D cultivation and in vivo animal F I G U R E 4 Effects of FSTL1 on IL-1β, TNF-α and IL-6 expression in chondrocytes. Each column represents the mean ± SD. FSTL1 (1, 5 and 25 ng/mL) significantly induced IL-1β, TNF-α and IL-6 mRNA levels, as determined by RT-PCR. Only the high dose (5 and 25 ng/mL) of FSTL1stimulated IL-1β, TNF-α and IL-6 protein expression, as determined by ELISA. *P < 0.05 vs control group F I G U R E 5 Effects of FSTL1 on nuclear factor kappa B (NF-κB) activation in chondrocytes. FSTL1 (1, 5 and 25 ng/ mL) induced the degradation of IκBα by simulating its phosphorylation. FSTL1 also significantly increased the level of p-p65. GAPDH was used as the endogenous control. *P < 0.05 vs control group OA model, should be carried out to evidence its phenotype and mechanism.
In conclusion, our study demonstrated that FSTL1 significantly increases the mRNA and protein levels of MMP-1, MMP-13, COX-2, iNOS, TNF-α, IL-1β and IL-6 in chondrocytes. This pro-inflammatory effect can be attributed to an increase in NF-κB activity. FSTL1 may therefore be a therapeutic target in OA.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China (grant no. 81301584).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.