NAV2 positively modulates inflammatory response of fibroblast‐like synoviocytes through activating Wnt/β‐catenin signaling pathway in rheumatoid arthritis

Abstract

Dear Editor, Rheumatoid arthritis (RA) is a common chronic autoimmune disease that causes progressive joint destruction and involves severe damage to physical function and life quality.1 Without rational drug intervention, 80% patients will become incapable of mobility after 3 years of illness.2 In the pathogenesis of RA, activated fibroblast-like synoviocytes (FLS) participate in the inflammatory process of RA through their own proliferation, migration, and invasion.3, 4 However, despite the thorough research, there are still gaps in our understanding of RA pathogenesis. Therefore, it is important to better understand the etiopathogenesis of RA. Neuron navigator 2 (NAV2) belongs to the neuron navigator family which mainly regulates growth, migration, and regeneration of neurons in the nervous system and is highly expressed in the brain, kidney, liver, and also existed in the skeleton, heart, placenta, as well as abundantly elevated in colon cancers.5 Previous studies have found that NAV2 facilitates invasion of cutaneous melanoma cells and plays a vital role in the poor prognosis of melanoma patients.6 Also, in the results of proteomics, NAV2 is abnormally highly expressed in RA patients’ primary FLS compared with healthy volunteers.7 However, the specific role of NAV2 in RA remains unknown. We first examined NAV2 protein level in blood samples and found a significant increase of NAV2 expression in RA patients compared with osteoarthritis (OA) patients or healthy samples (Figure 1A). The clinically relevant information and spreadsheet are shown in Table S1. Then we performed an immunofluorescence double-staining experiment. The expression of vimentin indicated that the cells derived from synovium tissues were primary FLS and NAV2 expression was significantly upregulated (Figure 1B). Moreover, we confirmed the significant elevation of NAV2 in the primary RA synovial cells by using RT-qPCR and Western blot analysis (Figure 1C). Then we observed and validated NAV2 expression in AIA rat synovial tissues. Images of rats’ hind paws showed significant exacerbation in inflammation and soft tissue swelling. Micro-CT and histological analysis presented swollen joints, bone destruction, synovial membrane hyperplasia accompanying serious inflammatory cell infiltration, and joint tissues with pannus formation (Figure S1A). The model group also developed much more severity and higher incidence of arthritis as determined by arthritis scores and hind paw volumes compared to the normal group, and the mean body weights of rats in the two groups increased during the experiment (Figure S1B). We furthermore monitored the production of proinflammatory cytokines IL-Iβ, IL-6, and TNF-α in the serum by ELISA kit, as shown in Figure S2A, the levels of IL-Iβ, IL-6 and TNF-α were dramatically increased. Additionally, the protein expression of MMP-9, COX-2, and IL-6 in inflamed joints was significantly upregulated (Figure S2B), indicating that the AIA model has been successfully established. Intriguingly, expression of NAV2 markedly increased both on the protein level and mRNA level (Figure 1D). And we could see the average integrated optical density (IOD) of NAV2 protein was significantly higher in the ankle joint of AIA rats (Figure 1E). In vitro assay, first, we used MH7A cells treated with TNF-α (20 ng/ml) for 0, 3, 6, 12, 24 and 48 h to evaluate an inflammatory response (Figure S3A-G). Interestingly, NAV2 mRNA level reached a peak at 3 h and then declined, NAV2 protein level showed a continuous increase from 0 to 24 h and decreased slightly at 48 h (Figure 2A). After transfection with siRNA to knockdown NAV2 expression in MH7A cells, we could see that siRNA downregulated NAV2 mRNA and protein expression when compared with scrambled control siRNA (si Scr) (Figure 2B). Intriguingly, accompanied by a decrease of NAV2, the protein and mRNA expression of IL-6, MMP-9, and COX-2 were also decreased (Figure 2C-E). Besides, silencing NAV2 significantly decreased protein expression of ICAM-1 and VCAM-1 (Figure 2F and G). Also, the mRNA level of IL-8 declined after NAV2 knockdown (Figure 2H). More importantly, lower NAV2 expression led to the decline of IL-1β and IL-6 secretion in the supernatants of the TNF-α-stimulated MH7A cells (Figure 2I and J). Immunofluorescence double staining also displayed that the co-expressions of NAV2 and COX-2 upon TNF-α induction disappeared when NAV2 was knocked down (Figure 2K). As Wnt/β-catenin pathway was stimulated in AIA rats (Figure S2C), the protein levels of GSK-3β, β-catenin, c-myc, CyclinD1, and MMP-3 appeared to be markedly attenuated when NAV2 was silenced (Figure 2L). Furthermore, silenced NAV2 expression significantly impeded cell motility, migration and invasive capabilities. Cell growth rates in si NAV2-transfected cells were also markedly diminished (Figure 2 M). We next investigated the underlying mechanisms by which NAV2 promoted inflammatory response in RA. We found that NAV2 could be regulated by transcription factor E2F1. First, we found E2F1 increased in RA FLS and AIA rat synovial tissues (Figure 3A and B), as well as in MH7A cells subjected to TNF-α for indicated times (Figure 3C). Then overexpression of E2F1 promoted NAV2 protein level, while knockdown of E2F1 by siRNA inhibited NAV2 expression. Also, we found that overexpression of E2F1 upregulated MMP-3 and MMP-9 expression, which are the downstream proteins in Wnt/β-catenin signaling pathway and related with invasion and proliferation in RA, and also increased phenotype protein levels of VCAM-1 and ICAM-1 significantly. Whereas knockdown of E2F1 produced the opposite effect (Figure 3D-O). Immunofluorescence double analysis confirmed that treatment with si E2F1 also resulted in decreased E2F1 and NAV2 significantly (Figure 3P). Furthermore, we found that elevated E2F1 expression could enhance NAV2 transcription level dramatically by luciferase reporter assay (Figure 3Q). Then the CHIP-qPCR results indicated that E2F1 could bind to the promoter region of NAV2 from -2000 to +500 (Figure 3R and S). In conclusion, the present study revealed an important novel finding that NAV2 plays a critical role in RA, and provided a new understanding of the molecular mechanism of RA which is unique and previously unreported. We also speculate targeting NAV2 might not just affect inflammation in RA but could also interfere with a major cell-cell interaction involved in sensitization of joint-innervating neurons that drive pain in arthritis.8 Therefore, NAV2 provides an attractive novel target to intervene inflammatory diseases, especially RA. This work was supported by grants from Macau Science and Technology Development Fund (FDCT) (033/2017/AMJ, 067/2018/A2, 0007/2019/AKP, and 0052/2020/A). R. Wang, J. Mao, and Y. Zhu designed research, analyzed data, and wrote the manuscript; and R. Wang, M. Li, W. Wu, Y. Qiu, W. Hu, Z. Li, Z. Wang, Y. Yu, J. Liao, W. Su performed research. All authors read and approved the manuscript. All the experimental processes were conducted within the approved guidelines of the Ethics Review Committee for Animal Research of Macau University of Science & Technology. Appropriate measures were taken to minimize the use of animals as well as their suffering. The authors declare that they have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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