Knockdown of nrf2 Exacerbates TNF-α-Induced Proliferation and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes through Activating JNK Pathway

Fibroblast-like synoviocytes (FLS) in the synovial tissue of rheumatoid arthritis (RA) exhibit over-proliferative and aggressive phenotypes, which participate in the pathophysiology of RA. In RA, little is known about the nonantioxidant effect of nuclear factor erythroid 2-related factor 2 (nrf2), the master regulator of redox homeostasis. In this study, we aimed to explore the expression and upstream regulatory factors of nrf2 and revealed its functions in modulating the proliferation and invasion in RA-FLS. FLS were isolated from RA and osteoarthritis patients. Expression of nrf2 in the synovial tissues and FLS was analyzed by immunohistochemistry, real-time PCR, Western blotting, and immunofluorescence staining. Cell proliferation was examined by Cell Counting Kit-8. Cell invasion was tested by transwell assay. Phosphorylation of JNK was determined by Western blotting. The results showed that nrf2 expression in the RA synovial tissues was upregulated. TNF-α promoted expression and nuclear translocation of nrf2 in RA-FLS and increased the intracellular reactive oxygen species (ROS) level. Nrf2 nuclear translocation was blocked by ROS inhibitor N-acetylcysteine. Both knockdown of nrf2 by siRNA and inhibition of nrf2 by ML385 significantly promoted the TNF-α-induced proliferation and invasion of RA-FLS. Activation of nrf2 by sulforaphane (SFN) profoundly inhibited the TNF-α-induced proliferation and invasion of RA-FLS. Knockdown of nrf2 also enhanced the TNF-α-induced matrix metalloproteinases (MMPs) expression and phosphorylation of JNK in RA-FLS. Proliferation and invasion of RA-FLS incubated with TNF-α and nrf2 siRNA were inhibited by pretreatment with JNK inhibitor SP600125. In conclusion, nrf2 is overexpressed in synovial tissues of RA patients, which may be promoted by TNF-α and ROS levels. Activation of nrf2 may suppress TNF-α-induced proliferation, invasion, and MMPs expression in RA-FLS by inhibiting JNK activation, indicating that nrf2 plays a protective role in relieving the severity of synovitis in RA. Our results might provide novel insights into the treatment of RA.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the joints, characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage and bone destruction, which would ultimately lead to irreversible functional loss [1]. Although major progress has been made in RA treatment, there are still concerns about incomplete responses and toxicity. Further elucidation of RA pathogenesis and the development of effective therapies remain an urgent need.
Synovium comprises an intimal lining layer and a sublining layer, where two major cell types are located: fibroblastlike synoviocytes (FLS) and macrophage-like synoviocytes. Normal synovium has a thin intimal lining layer consisting of two to three layers of cells; however, the intimal lining layer in RA becomes inflamed, hyperplastic with expansion in both cell types, and invasive of cartilage and bone [2,3].
RA-FLS exhibits an aggressive tumor-like phenotype that increases the invasiveness into the extracellular matrix (ECM) and further aggravates the joint damage [4]. However, the mechanisms underlying the abnormal growth and invasion of RA-FLS are not fully understood.
Oxidative stress occurs in RA due to an imbalance in the generation and removal of reactive oxygen species (ROS), leading to inflammation, abnormal cellular behavior, and tissue damage [5]. Nuclear factor erythroid 2-related factor 2 (nrf2) as a transcription factor and a master regulator of antioxidant response has been considered a therapeutic target for RA [6]. Under basal conditions, nrf2 binds to the inhibitory Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm, which targets nrf2 to proteasomal degradation [7]. When being activated by ROS or electrophilic compounds such as sulforaphane (SFN), nrf2 translocates into the nucleus after being released from Keap1 and subsequently regulates the expression of targeted genes [8][9][10].
The transactivation of multiple genes involved in the antioxidant response, immune process, and metabolism by nrf2 has been well recognized [9]. It was originally considered to exert protection against oxidative stress and inflammation in various diseases including RA. Nrf2 deficiency aggravated disease severity and inflammatory condition in RA animal models [11,12]. Chemicals that directly activate nrf2 showed anti-inflammatory and immunoregulatory effects [6,13] Nrf2 also mediated the therapeutic effects of many drugs in RA, such as licochalcone A and resveratrol [14][15][16]. Nevertheless, recent studies noted that nrf2 could also play a role in cancer progression, promoting proliferation and invasion in glioblastoma and hepatocellular carcinoma [17][18][19]. These remind us that nrf2 might modulate RA disease severity through other mechanisms such as controlling cell growth and invasion. Upregulated nrf2 has been found in the FLS of RA patients, but how nrf2 modulates the proliferation and invasion of RA-FLS remains unknown [11].
Here, we confirmed the higher expression of nrf2 in the RA synovium and revealed that TNF-α contributed to the activation of nrf2 by inducing intracellular ROS in RA-FLS. More importantly, we identified knockdown of nrf2 as an enhancer of proliferation, invasion, and the expression of the matrix metalloproteinases (MMPs) of RA-FLS by activating the JNK pathway.

Materials and Methods
2.1. Tissue Samples. Synovial tissues from RA and osteoarthritis (OA) patients were obtained during the total knee arthroplasty. Patients were diagnosed according to the 2010 ACR/EULAR classification criteria for RA or 1986 revised ACR classification criteria for OA [20,21]. This study complied with the Declaration of Helsinki (1964), and the research protocol was approved by the Ethic Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Informed consent was obtained from all the patients for tissue donation.
2.2. Isolation and Culture of Human FLS. Synovial tissues were used to isolate primary FLS. The fat, fibrous mem-branes, and cartilage fragments were removed. The synovial tissue was minced into fragments and digested with 1 mg/mL type II collagenase (Merck Millipore, USA) dissolved in Dulbecco's Modified Eagle Medium (DMEM) at 37°C overnight. After that, the digested mixture was pipette through a 70 μm mesh cell filter (Merck Millipore, USA) into a 50 mL tube and centrifuged at 250 g for 10 min. The supernatant was removed, and the cell pellet was washed with DMEM three times. Finally, the cell pellet was resuspended with DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (Gibco, USA). The cells were cultured in 10 cm culture dishes at a density of 2:5 × 10 4 cells/mm 2 in a humidified incubator (Thermo Fisher Scientific, USA) containing 95% air and 5% CO 2 at 37°C and subcultured upon confluence. Passages 3-8 were used for experiments.

Immunohistochemistry and Immunofluorescence Staining.
Synovial tissues were fixed in 4% paraformaldehyde (PFA) for 48 h and embedded in paraffin. Serial sections (5 μm) were made for immunohistochemistry and hematoxylin and eosin (HE) staining. For immunohistochemistry, synovial tissue sections were deparaffinized with xylene and rehydrated with gradient ethanol. Antigen retrieval was conducted with citrate buffer at 95°C for 8 min. For immunofluorescence staining, cells were fixed with 4% PFA for 10 min and permeabilized with 0.25% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. Tissue sections or cells were blocked with 10% donkey serum (Jackson ImmunoResearch Labs, USA) in tris-buffered saline (TBS) for 1 h at room temperature and incubated with anti-nrf2 antibodies (Abcam, UK, 1: 500) or rabbit IgG (Merck Millipore, USA, 1 : 500) at 4°C overnight. Then, they were washed with TBS (0.025% Triton X-100) for 2 × 5 min. Next, tissue sections were incubated with horseradish peroxidase-(HRP-) conjugated donkey anti-rabbit IgG (Jackson Immu-noResearch Labs, USA, 1 : 1000) at room temperature for 1 h and stained with diaminobenzidine (DAB, Beyotime Biotechnology, China) to visualize the staining. Cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody (Abcam, UK, 1 : 200) in darkness for 30 min at room temperature. Cells were mounted with mounting media containing DAPI (4 ′ ,6-diamidino-2-phenylindole) (Beyotime Biotechnology, China). Digital images were captured from synovial tissue sections through an optical microscope (Olympus, Japan) and from cells with a fluorescence microscope (Olympus, Japan).

Results
3.1. Upregulation of nrf2 in the Synovial Tissues of RA Patients. Immunohistochemistry staining was performed to determine the nrf2 protein expression level in the synovial tissues from RA and OA patients using anti-nrf2 antibodies and rabbit IgG (isotype). Nrf2 expression was significantly upregulated in the RA synovium compared with OA; synovial hyperplasia was also observed in RA with HE staining (Figure 1).

Induction of nrf2 Expression and Nuclear
Translocation by TNF-α through Inducing ROS. TNF-α is one of the most important mediators of RA synovitis. To investigate whether TNF-α could cause nrf2 upregulation and activation, RA-FLS were treated with TNF-α (25 ng/mL). The upregulated expression of nrf2 mRNA was demonstrated after 3, 6, and 24 h of TNF-α treatment with real-time PCR analysis (Figure 2(a)). The nuclear protein level of nrf2 was increased in RA-FLS after stimulation with TNF-α (25 ng/mL) at different time points (Figures 2(b) and 2(c)), indicating that TNFα could promote nrf2 nuclear translocation. Furthermore, the nuclear translocation of nrf2 after TNF-α (25 ng/mL) treatment was confirmed with immunofluorescence staining (Figure 2(d)).
To illustrate whether ROS mediates TNF-α-induced nrf2 activation, the intracellular ROS level of RA-FLS was 3 Journal of Immunology Research detected. TBHP was used as a positive control. It was found that TNF-α (25 ng/mL) enhanced intracellular ROS levels in a time-dependent manner (Figure 2(e)). Further treatment with ROS inhibitor N-acetylcysteine (NAC) blocked TNF-αinduced nrf2 nuclear translocation (Figure 2(f)).

Enhancement of Proliferation and Invasion by
Knockdown of nrf2 in RA-FLS. Next, the biological effects of nrf2 in RA-FLS were explored. Successful nrf2 knockdown by siRNA was confirmed by real-time PCR and Western blotting (Figures 3(a)-3(c)). The effects of knockdown of nrf2 on the TNF-α-induced proliferation rate and invasion in RA-FLS were analyzed with CCK-8 and transwell assay. As a result, TNF-α (25 ng/mL) promoted the proliferation and invasion of RA-FLS, which were further enhanced by knockdown of nrf2 (Figures 3(d)-3(f)).

Discussion
The present study demonstrated that TNF-α promoted the nrf2 expression and nuclear translocation by upregulating intracellular ROS level in RA-FLS. Silencing nrf2 further facilitated the TNF-α-induced RA-FLS proliferation and invasion by promoting the expression of MMPs partly through activating the JNK pathway. These results indicate that nrf2 acts as a negative-feedback regulator in the RA-FLS against TNF-αinduced proliferation, invasion, and MMP expression. It plays a protective role in relieving the severity of synovitis in RA.  First, nrf2 protein expression was verified in the human synovial tissues. Nrf2 expression in RA synovial tissues was much higher than OA, which was consistent with the previous study where Wruck et al. [11] demonstrated that nrf2 was activated in RA synovium compared with healthy donors. Proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, play pivotal roles in the pathogenesis of RA. TNF-α is one of the most established effectors that mediate synovial inflammation and imbalanced systemic immune status [23]. In monocytes, TNF-α induced sustained nrf2 activation and increased expression of nrf2 targeted genes [24]. Besides, a low concentration of TNF-α evoked significant nuclear translocation of nrf2 and transactivation of nrf2 targets [25]. Here, it was found that TNF-α not only upregulated nrf2 expression but also triggered nrf2 nuclear translocation, indicating that increased TNF-α level in RA patients might at least partly account for the nrf2 overexpression in the RA synovium. Then, the mechanisms that mediated the TNF-α-induced nrf2 activation were explored. It has been well recognized that intracellular ROS and electrophiles are potent nrf2 activators by modulating the conformation of the nrf2 adaptor keap1 and stop the nrf2 from  Journal of Immunology Research going through keap1-dependent ubiquitination and proteasomal degradation [9]. TNF-α could increase ROS production in mouse embryonic fibroblasts, Hela cells, and murine hepatocytes [26][27][28]. As expected, in the present study, TNF-α induced ROS production in RA-FLS, and pretreatment with ROS scavenger NAC inhibited the TNF-α-induced nrf2 nuclear translocation, indicating that ROS mediated the TNF-α-induced nrf2 activation in RA-FLS. Despite the widely studied powerful antioxidant ability, nrf2 exerts multiple nonantioxidant effects. Recent studies addressed that nrf2 modulated cell proliferation, survival, and invasion, although the modulating direction depended on different cellular microenvironments. Nrf2 deficiency disrupted self-renewal of the airway basal stem cells [29]. Knockout of nrf2 delayed proliferation of hepatocytes after hepatectomy [30]. Nevertheless, type 2 innate lymphoid cells from nrf2-deficient mice underwent hyperproliferation in response to the stimulation of IL-33 combined with IL-2, IL-7, or thymic stromal lymphoprotein [31]. ML385 is a probe molecule that binds to the Neh1 domain of nrf2 and interferes with the binding of V-maf avian musculoaponeurotic fibrosarcoma oncogene homologue G with nrf2 and blocks the nrf2 targeted genes expression [22]. Here, both knockdown of nrf2 by siRNA and inhibiting nrf2 by ML385 significantly promoted the TNF-α-induced proliferation of RA-FLS. SFN is the bioactive form of glucoraphanin that is mostly found in cruciferous vegetables; it effectively activates nrf2 by modifying the sensor cysteine in keap1 [32]. The present study further demonstrated that activating nrf2 by SFN profoundly inhibited the TNF-α-induced proliferation of RA-FLS. All these data suggest that increased and activated nrf2 secondary to inflammatory microenvironment may counteract the hypertrophy of RA synovium. The invasive characteristic of RA-FLS is one of the major causes of bone and cartilage destruction in RA. So far, how nrf2 modulates the invasion of RA-FLS has not been elucidated. Most studies regarding the effect of nrf2 on cell invasion were conducted in cancer cells, and the results were paradoxical. Overexpression of nrf2 in oral squamous cell carcinoma cells promoted cell migration and invasion [33]. However, stabilizing nrf2 in breast cancer cells inhibited invasion and expression of recepteur d'origine nantais, a tyrosine kinase receptor that promotes cancer cell invasion [34]. The present study showed that both silencing nrf2 and inhibiting nrf2 by ML385 promoted the invasion of RA-FLS. On the contrary, activating nrf2 by SFN inhibited the TNF-α-induced invasion of RA-FLS. These results further suggest that nrf2, being a negative feedback regulator against TNF-α-induced synovitis in RA, might exert a protective effect by downregulating the invasiveness of RA-FLS.
Rheumatoid synoviocytes express a battery of MMPs that are the primary effectors responsible for pathologic ECM remodeling and cell invasion in RA [35]. The expression levels of MMP1, MMP2, MMP3, MMP9, and MMP14 were tested, which are among the major types contributing to the process of joint destruction in RA [36,37]. It was found that knockdown of nrf2 could enhance the expression of these MMPs. Consistent with our results, Choi et al. revealed that nrf2 activator SFN inhibited IL-1β-induced expression of MMP-1, MMP-3 in RA-FLS [38]. Activation of nrf2 reduced UVA-mediated MMP1 upregulation in human keratinocyte cell line [39]. Moreover, disruption of nrf2 enhanced the MMP9 expression after spinal cord injury in mice [40]. In summary, the above data suggest that nrf2 may downregulate the invasiveness of RA-FLS by inhibition of MMP production.
The promoter regions of many MMPs contain the binding site of c-Fos/activator protein-1 (AP-1), which is critical for MMPs expression and locates downstream of the JNK MAPK and ERK MAPK kinases [41]. It has been well recognized that TNF-α regulates the expression of MMPs via the MAPK signaling cascades in RA-FLS [42]. Knockdown of nrf2 could also activate the MAPK pathways in UVradiated human keratinocytes [39,43]. Thus, the pathways involving JNK and ERK were analyzed in the present study. As a result, the phosphorylation of JNK was enhanced in the RA-FLS treated with TNF-α alone, and the upregulated phosphorylation of JNK was further enhanced by nrf2 siRNA. Besides, blocking JNK with its inhibitor SP600125 reduced not only the invasion but also the MMP9 expression mediated by knockdown of nrf2 in RA-FLS. In addition, several studies noted the proproliferative effect of JNK pathway [44,45], which was in line with the finding that the proliferation of RA-FLS incubated with TNF-α and nrf2 siRNA was inhibited by pretreatment with JNK inhibitor SP600125. These data suggest that nrf2 may inhibit the activation of JNK pathway to downregulate MMP expression, invasion, and proliferation of RA-FLS.

Conclusions
In conclusion, nrf2 is overexpressed in synovial tissues of RA patients, which may be promoted by TNF-α and ROS levels. Increased nrf2 may suppress TNF-α-induced proliferation, invasion, and MMPs expression in RA-FLS through inhibiting JNK activation. Our findings offer a novel insight into the protective role that nrf2 played in RA.

Data Availability
All data are available from the corresponding author on reasonable request.