CX3CL1 promotes MMP-3 production via the CX3CR1, c-Raf, MEK, ERK, and NF-κB signaling pathway in osteoarthritis synovial fibroblasts

Written informed consent was obtained from all patients recruited into this study, and the study was approved by the Institutional Review Board of Shin Kong Wu Ho-Su Memorial Hospital. Synovial tissue was obtained from patients with OA, and SFs were isolated. Human SFs were isolated by collagenase treatment of synovial tissue samples obtained from 10 patients with OA during knee-replacement surgeries and eight samples of nonarthritic synovial tissues obtained at arthroscopy after trauma/joint derangement. Fresh synovial tissues were finely minced and digested in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mg/ml type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37 °C and under 5% CO₂. The isolated cells were placed in DMEM containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine at 37 °C with 5% CO₂. Passages 4–6 of the obtained OASFs were used in this study. Results of four independent experiments are presented [4, 17].

DMEM, Lipofectamine3000, and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture dishes, FBS, six-well plates, and 12-well plates were purchased from Corning (Corning, NY, USA). Polyvinyldifluoride (PVDF) membranes and an Immobilon Western Chemiluminescent HRP Substrate detection system were purchased from Millipore (Billerica, MA, USA). Polyclonal antibodies specific for MMP3, CX3CL1, CX3CR1 and IKKα/β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies specific for c-Raf, MEK, ERK, IκBα, p65, and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit antibodies specific for c-Raf phosphorylated at Ser338 and IKKα/β phosphorylated at ser176/180 were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). Monoclonal rabbit antibodies specific for MEK1/2 phosphorylated at Ser217/221, ERK1/2 phosphorylated at Thr202/204, IκBα phosphorylated at ser32/36, and p65 phosphorylated at ser536 were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA).3-(3,5-Dibromo-4-hydroxybenzyliden)-5-iodo-1,3-dihydroindol-2-one (GW5074), 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene monoethanolate (U0126), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), pyrrolidine dithiocarbamate (PDTC), and l-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK) were purchased from Sigma-Aldrich. Recombinant human CX3CL1 was purchased from PeproTech (Rocky Hill, NJ, USA). The small interfering RNA (siRNA) of the control and CX3CR1 siRNA were purchased from Santa Cruz Biotechnology. Nuclear factor kappa B (NF-κB) luciferase plasmid was purchased from Stratagene (La Jolla, CA, USA). A pSV-β-galactosidase vector and a luciferase assay kit were purchased from Promega (Madison, MA, USA). All other chemicals were purchased from Sigma-Aldrich.

Total RNA was extracted from cells using Trizol reagent (Invitrogen) following the manufacturer’s protocol. In brief, cells were added to 0.5 ml Trizol, homogenized, and incubated at room temperature for 3 min. After extraction with chloroform (0.1 ml) and precipitation with isopropanol (0.4 ml), RNA was washed with 75% ethanol, and finally the RNA pellet was dissolved in 10 μl of RNase-free water. The RNA yield and purity were determined by measuring absorbance at 260 and 280 nm using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA was then used to synthesize complementary DNA (cDNA) using reverse transcriptase (Invitrogen) according to the manufacturer’s instructions.

Real-time quantitative polymerase chain reaction (qPCR) was performed using SYBR Green (KAPA Biosystems, Woburn, MA, USA) according to the manufacturer’s protocol, and reactions were performed using a StepOnePlus machine (Applied Biosystems, Foster City, CA, USA). Human MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) purchased from Sigma-Aldrich were used as primers to amplify the target genes. The expression levels of the target genes were determined by normalizing them to the GAPDH levels. We calculated the results using the following equation:

Each sample was assayed in triplicate, and the data represent three independent experiments.

Cellular lysates were prepared using the methods outlined in previous studies. Proteins were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immobilon PVDF membranes. The blots were blocked with 5% BSA for 1 h at room temperature and then probed using antihuman antibodies against MMP-3, CX3CL1, CX3CR1, c-Raf, MEK, ERK, IKKα/β, IκB, p65, and β-Actin (1:1000) for 1 h at room temperature. After three washes, the blots were incubated with secondary antibodies (1:10,000) for 1 h at room temperature. The blots were then visualized using a charge-coupled device camera-based detection system (UVP Inc., Upland, CA, USA). Quantitative data were obtained using ImageJ software (National Institute of Health, USA).

MMP-3 in the cell culture supernatants was then determined using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. In brief, OASFs were seeded in 100-mm culture dishes at a density of 5 × 10⁶ cells per dish and then treated under different conditions. Following 24 h of incubation, the culture supernatant was collected and centrifuged at 10,000 rpm for 10 min and stored at −80 °C in fresh tubes.

Transfection was performed using Lipofectamine 3000 transfection reagent (LF3000; Invitrogen) according to the manufacturer’s instructions. Cells were transfected with control siRNA, CX3CR1 siRNA, vector, dominant negative MEK mutants, dominant negative ERK mutants, dominant negative IKKα mutants, dominant negative IKKβ mutants, and luciferase plasmid using Lipofectamine 3000 in optiMEM medium. After 24 h of transfection, cells were incubated with the indicated agents. After 24 h of incubation, the luciferase activity in the transfected cells was measured using a Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Transactivation was determined by monitoring the firefly luciferase levels in the pGL2 vector. The luciferase assay was performed by adding lysis buffer (100 μl) and harvesting the cells through centrifugation (13,000 rpm for 5 min). The supernatant was transferred to fresh tubes, and 20 μl of cell lysate was added to 80 μl of fresh luciferase assay buffer in an assay tube. The luciferase activity was measured using a microplate luminometer. Luciferase activity was normalized to transfection efficiency based on the cotransfected β-galactosidase expression vector.

Crosslinked chromatin was prepared from OASF cells, and a chromatin immunoprecipitation (ChIP) assay was performed using a Pierce Magnetic ChIP kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. After immunoprecipitation with anti-p65 antibody or control IgG, protein A/G magnetic beads were added. DNA was purified and analyzed using PCR. The following MMP-3 primers were used: 5′-AATTCACATCACTGCCACCA-3′ (forward) and 5′-CTCTGTGGCAATAAGATCCC-3′ (reverse).

Values are reported as mean ± standard error of the mean (SEM). A statistical comparison between two samples was performed using the Student t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with the Bonferroni post-hoc test. In all comparisons, p < 0.05 was considered significant.

CX3CL1 is known to participate in the pathogenesis of OA and RA pathogenesis [14, 18]. Therefore, we first compared the CX3CL1 levels in normal human SFs (normal SFs) and OASFs. The mRNA expression of CX3CL1 was higher in the OASFs than in the normal SFs (Fig. 1a). Because CX3CL1 stimulates MMP expression in chronic liver diseases [19], we hypothesized that any of these MMPs could be involved in CX3CL1-directed OA pathogenesis. We used qPCR to detect mRNA expression levels of MMPs in normal SFs and OASFs. The expression of MMP-3 was significantly higher than that of other MMPs in OASFs compared with the basal level expressed in normal SFs (Fig. 1b, c). To understand the relationship between CX3CL1 and MMP-3 in normal SFs and OASFs, we examined the level of MMP-3 after CX3CL1 treatment. The level of MMP-3 was significantly elevated in OASFs compared with normal SFs. CX3CL1 induced MMP-3 production in a concentration-dependent manner (Fig. 1d–f), and induction occurred in a time-dependent manner in OASFs (Fig. 1 g–i). These results indicated that CX3CL1 increased MMP-3 production in human OASFs.

The CX3CL1–CX3CR1 axis plays a crucial role in the development of inflammatory diseases [10, 20]. Therefore, we hypothesized that CX3CR1 is involved in CX3CL1-induced MMP-3 production. We knocked down CX3CR1 expression by transfecting the OASFs with CX3CR1 siRNA and determined that CX3CR1 siRNA inhibited CX3CL1-induced MMP-3 production at the mRNA and protein expression levels (Fig. 2a, b). Furthermore, a CX3CL1 monoclonal antibody (mAb), but not the control IgG, effectively suppressed CX3CL1-induced MMP-3 mRNA and protein expression (Fig. 2c–e). These results suggest that CX3CR1 activation may be responsible for CX3CL1-induced MMP-3 expression.

To examine the mechanism by which CX3CL1 induces MMP-3 production, we directly measured the c-Raf phosphorylation in response to CX3CL1. The results revealed that the stimulation of cells using CX3CL1 induced c-Raf phosphorylation in a time-dependent manner (Fig. 3a). Pretreatment of cells with the CX3CR1 antibody attenuated c-Raf phosphorylation, suggesting that CX3CR1 serves as the upstream regulator of c-Raf-mediated signaling (Fig. 3b). Furthermore, to examine whether CX3CL1 stimulates the production of MMP-3 through c-Raf signaling, we used pharmacological inhibitor (GW5704) and c-Raf shRNA. Pretreatment of cells with GW5074 was found to antagonize CX3CL1-induced MMP-3 production at the mRNA and protein levels (Fig. 3c–e). As shown in Fig. 3f, g, CX3CL1-induced MMP-3 production at the mRNA and protein levels was strongly reduced in shRNA against c-Raf.

Subsequently, we investigated whether CX3CL1 can activate MEK/ERK, which is a critical downstream target of c-Raf. Stimulation of cells with CX3CL1 was discovered to induce the time-dependent phosphorylation of MEK and ERK (Fig. 4a). However, this CX3CL1-induced phosphorylation of MEK/ERK was markedly decreased by inhibiting upstream signaling events using the c-Raf inhibitor (GW5704) (Fig. 4b). To further evaluate whether the MEK/ERK pathway can induce MMP-3 expression, we pretreated cells with PD98059 (10 μM) and U0126 (10 μM). CX3CL1 induced the mRNA and significantly reduced the protein levels of MMP-3 when cells were pretreated with PD98059 and U0126 (Fig. 4c–e). To further confirm this stimulation-specific mediation by MEK and ERK, we assessed the role of MEK and ERK using dominant negative mutations. Transfection of cells with dominant negative MEK and dominant negative ERK reduced MEK and ERK expression, respectively (Fig. 4f, upper panel). Transfection of cells with dominant negative MEK and dominant negative ERK effectively inhibited CX3CL1-induced MMP-3 mRNA and protein expression (Fig. 4e–g). These results indicate that CX3CL1 induces MMP-3 production through CX3CR1 activation, which consequently activates the c-Raf/MEK/ERK signaling pathways in OASFs.

The activation of NF-κB can induce MMP-3 production in the cells of patients with RA or OA [21]. NF-κB is a transcriptional activator that plays a vital role in OA pathogenesis [22]. To examine whether NF-κB is involved in the signal transduction pathway leading to CX3CL1-induced MMP-3 production, we used NF-κB inhibitors (PDTC) and IκB protease inhibitors (TPCK). Pretreatment of cells with PDTC and TPCK was discovered to inhibit CX3CL1-induced MMP-3 mRNA and protein expression (Fig. 5a–c). We further examined the upstream molecules involved in CX3CL1-induced NF-κB activation. The stimulation of OASFs using CX3CL1 increased IKKα/β, IkBα, and p65 phosphorylation in a time-dependent manner (Fig. 5d). In addition, transfection of cells with IKKα and IKKβ mutants reduced CX3CL1-induced MMP-3 production and MMP-3 mRNA expression (Fig. 5e, f).

To confirm that NF-κB is involved in CX3CL1-induced MMP-3 expression, we performed transient transfection using NF-κB promoter–luciferase constructs. When OASFs were incubated with CX3CL1, the NF-κB promoter activity increased in a dose-dependent manner (Fig. 6a). The increase in NF-κB activity induced by CX3CL1 was antagonized by c-Raf inhibitor (GW5704), MEK inhibitors (PD98059 and U0126), and c-Raf shRNA, MEK, ERK, IKKα, and IKKβ mutants (Fig. 6b, c). Furthermore, GW5704, PD98059, and U0126 reduced CX3CL1-mediated p65 phosphorylation (Fig. 6d). In addition, these inhibitors (Gw5074, PD98059, and U0126) reduced the CX3CL1-induced binding of p65 to an NF-κB element (Fig. 6e). To further investigate CX3CL1-mediated MMP-3 expression in OASFs, we established CX3CL1-shRNA expression cells. Western blot analyses were employed to compare the CX3CL1 expression levels in stable transfectants. CX3CL1 expression was drastically inhibited in OASF/CX3CL1-shRNA cells (Fig. 6f–h). In addition, CX3CL1 knockdown downregulated the expression of MMP-3 in OASFs (Fig. 6f–h). These data suggest that the CX3CR1, c-Raf, MEK, ERK, and NF-κB pathways must be activated if CX3CL1-induced MMP-3 production is to occur in human OASFs.