Activity of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis Was Impaired by Dickkopf-1 Targeting siRNA

Activity of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis Was Impaired by Dickkopf-1 Targeting siRNA

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease that primarily affects the peripheral joints, leading to articular destruction and functional disability. Chronic synovitis, characterized by the proliferation of synovial lining cells, is a hallmark of RA. These cells are a significant source of chemokines and cytokines associated with inflammation, which can result in the destruction of articular cartilage and bone tissues. Fibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play a crucial role in the pathogenesis of RA. Activated FLSs produce a broad array of cell surface and soluble mediators that recruit, retain, and activate immune cells, leading to the dysregulated expression of inflammatory cytokines.

The Wnt signaling pathway is a key regulator of joint remodeling. Dickkopf-1 (DKK-1), an endogenous secreted factor, acts as an antagonist of the Wnt pathway by binding the Wnt co-receptor LRP5/6. DKK-1 is secreted by FLSs in response to inflammation and has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Previous studies have shown that serum levels of DKK-1 are significantly increased in patients with RA compared to healthy controls or individuals with other rheumatic diseases such as osteoarthritis and ankylosing spondylitis. Blockade of DKK-1 with an anti-DKK-1 antibody has been reported to reverse the bone-destructive pattern in experimental arthritis and mediate a bone-forming pattern. However, it is unknown whether silencing DKK-1 with small interfering RNAs (siRNAs) has the same effect as using an anti-DKK-1 antibody.

In this study, we investigated the effect of silencing DKK-1 by siRNAs in the FLSs of RA. We hypothesized that DKK-1 may serve as a therapeutic target in RA. To test this hypothesis, we isolated FLSs from synovial tissues obtained from patients with RA during knee replacement surgery. The synovial tissues were washed, minced, and incubated with collagenase to obtain a cell suspension. The cells were then cultured in RPMI 1640 medium supplemented with fetal bovine serum (FBS), penicillin, and streptomycin. Non-adherent cells, including macrophage-like cells and impurities, were removed by washing, and the FLSs were purified through passage.

To determine the effect of DKK-1 silencing, siRNAs targeting DKK-1 were transfected into FLSs using Lipofectamine 2000. Six siRNA duplexes targeting DKK-1 (D1–D6) were synthesized, and the most effective siRNAs were selected based on their ability to inhibit DKK-1 expression at both the mRNA and protein levels. The expression levels of DKK-1 in FLSs were detected by real-time quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The levels of interleukin (IL)-1b, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-b1, TGF-b2, and monocyte chemoattractant protein (MCP)-1 in the cell culture supernatant were also measured by ELISA. The effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation were investigated using invasion assays and 3H-thymidine incorporation assays, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-kB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK), and b-catenin in FLSs.

The results showed that siRNAs targeting DKK-1 significantly inhibited the expression of DKK-1 in FLSs. The most effective siRNAs, D2 and D6, were selected for subsequent experiments. DKK-1 targeting siRNAs induced a significant reduction in the levels of IL-6, IL-8, MMP2, MMP3, and MMP9 in FLSs compared to the control group. Additionally, siRNAs targeting DKK-1 inhibited the proliferation and invasion of FLSs. Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, b-catenin, a pivotal downstream molecule of the Wnt signaling pathway, was increased.

TNF-a is a major pro-inflammatory factor involved in the pathogenesis of RA. In our study, we mimicked the inflammatory environment of RA in vitro with TNF-a. We showed that TNF-a could stimulate FLSs to produce IL-6, IL-8, and MMPs, and promote proliferation and invasion of FLSs. siRNAs targeting DKK-1 inhibited the activity of FLSs with or without the stimulation of TNF-a. However, the anti-DKK-1 monoclonal antibody did not show the same effect as the one observed with siRNAs.

The invasive property of RA patient-derived FLSs has been shown to correlate with disease severity and radiographic damage. MMPs are key mediators of the invasive phenotype of FLSs. Our results show that reduced expression of DKK-1 in FLSs can decrease the production of MMPs and inhibit the invasive behavior of FLSs. Thus, siRNAs targeting DKK-1 may directly block the bone destruction of RA.

Another major consequence of RA FLSs activation is the synthesis of a broad array of soluble and cell surface pro-inflammatory molecules with many diverse functions through their activation of multiple signal transduction pathways, including NF-kB and mitogen-activated protein kinase (MAPK). Among the MAPK families, ERK, JNK, and p38 have been implicated in RA. JNK and ERK play very important roles in extracellular matrix turnover and are activated in the RA synovium. They regulate MMP gene expression in cultured FLSs and mediate joint destruction in rat adjuvant arthritis. DKK-1 siRNAs can significantly decrease the expression of ERK and MMPs in FLSs, suggesting that siRNAs targeting DKK-1 may improve FLSs features through the MAPK pathway.

Previous studies have demonstrated that the IL-1 receptor-associated kinase 1 (IRAK-1) suppresses IL-6/IL-8 secretion. Physiological studies have shown that Irak1-null mice are protected from various inflammatory diseases including experimental autoimmune encephalomyelitis and atherosclerosis, though the mechanism for this remains unknown. In this study, we found that siRNAs targeting DKK-1 down-regulated the secretion of IL-6 and IL-8, which may be mediated by the repression of IRAK-1 signaling.

Proteins synthesized by Wnt genes are key mediators of osteoblastogenesis and govern the formation of the skeleton during the development of the embryo. Several members of the Wnt protein family bind a receptor complex consisting of LPR5/6 and frizzled receptors on the plasma membrane of mesenchymal cells, which signals osteoblast differentiation by engaging the intracellular protein b-catenin. DKK-1 functions as a natural inhibitor of the Wnt signaling. In this study, the interference of DKK-1 with either siRNAs or an antibody targeting DKK-1 may increase the expression of b-catenin.

The limitations of this study are as follows: in vivo experiments could not be performed due to technical difficulties. The use of siRNA for in vivo applications remains difficult as optimizing the intracellular delivery of the siRNA and determining the stability of the compound pose complications. Ideally, these experiments may be repeated in vivo once some of these complications have been resolved.

In conclusion, siRNAs targeting DKK-1 exerted a profound inhibitory effect on the proliferation, invasive behavior, and inflammatory responses of FLSs, which was mediated by the ERK or the IRAK-1 signaling pathway. These data suggest that DKK-1 silencing represents a potential therapeutic approach for RA.

doi.org/10.1097/CM9.0000000000000697

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