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Akt and JNK signaling pathways
Chinese Journal of Natural Medicines 2015, 13(11): 08310841
Chinese Journal of Natural Medicines
Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways LV Qi, ZHU Xian-Yang △, XIA Yu-Feng, DAI Yue *, WEI Zhi-Feng * Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China Available online 20 Nov., 2015
[ABSTRACT] Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L−1) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. [KEY WORDS] Tetrandrine; Rheumatoid arthritis; Fibroblast-like synoviocytes; Migration; Invasion
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)11-0831-11
Introduction Rheumatoid arthritis (RA) is characterized by chronic
[Received on] 16-May-2015 [Research funding] This work was partially supported by the National Natural Science Foundation of China (No. 81373426) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. [*Corresponding author] Tel: +86-25-83271400, Fax: +86-2585301528, E-mail: [email protected] (WEI Zhi-Feng); yuedaicpu@ hotmail.com (DAI Yue) △ Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
synovitis, which causes synovial hyperplasia and ultimate joint damage [1-3]. Fibroblast-like synoviocytes (FLS), located at synovial intimal edge, are identified as key players in the pathophysiological process of RA [4]. There are reports indicating that RA-FLS share many similar properties with tumor cells: undergoing tumor-like proliferation, migration and invasion, as well as possessing increased resistance to apoptosis [5]. They can migrate to the unaffected joints, attach to cartilage and bone, and invade into the extracellular matrix [6]. All these findings suggest that FLS are important effectors for spreading arthritis destruction to distant joints. Migration and invasion are complex processes that need dynamic interactions between cells and the surrounding matrix, and coordinated programs executed by multiple pathways that integrate signals from the extracellular environment and remodeling the actin cytoskeleton [7]. It is
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well established that pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α which are deeply involved in the pathogenesis of RA, constitute key mediators of RA-FLS migration and invasion [8]. Cytokines, as activators of FLS, lead to activation of Rho GTPases and signaling pathways (including NF-κB, MAPK and PI3K/Akt) by binding with specific acceptors, subsequently up-regulating the expressions of effector proteins such as degrading enzyme MMP-9 and F-actin, and then mediating the migration and invasion of cells [9-10]. Targeted modulation of the signaling pathways that control the expressions of migration- and invasion-related proteins may be helpful for the intervention of RA. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, has been clinically used for the management of RA-related diseases in China for a long time. By oral administration, Tet could ameliorate adjuvant-induced arthritis in rats, a well-established animal model of human RA [11]. However, its mechanism remains to be elucidated. Recent evidence indicates that Tet could inhibit migration and invasion of various kinds of tumor cells such as human prostate cancer DU145 and PC-3 cells and lung cancer A549 cells [12-13]. Those findings attracted us to explore the anti-RA mechanism of Tet based on migration and invasion of FLS in the present study.
Materials and Methods Chemicals and reagents Tet (purity > 95%) was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). IL-1β was purchased from PeproTech Int. (Rocky Hill, CT, USA). Dulbecco’s modified eagle medium (DMEM) was purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Wisent Corporation (Nanjing, China). 24-well transwell inserts were purchased from Milllpore (Massachusetts, Billerica, USA). Cell-Light EdU Apollo 567 DNA kit was purchased from RiboBio Co. Ltd. (Guangzhou, China). Monoclonal antibodies against GAPDH, MMP- 2/9, p38, p-p38, JNK, p-JNK, ERK, p-ERK, Akt, p-Akt, F-actin, FAK, p-FAK, Rac1, Cdc42, and RhoA were purchased from Bioworld (Minneapolis, MN, USA). SB203580 (a specific inhibitor of p38 MAPK), U0126 (a specific inhibitor of ERK), SP600125 (a specific inhibitor of JNK), LY294002 (a specific inhibitor of PI3K), and Triciribine (a specific inhibitor of Akt) were purchased from KangChen Bio-tech (Shanghai, China). The other chemicals and reagents were of analytical grade and obtained from commercial sources. Cell cultures The synovial tissues of RA patients were collected during knee joint arthroscopy according to the protocol approved by Institutional Ethics Review Committee of the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. The tissues were minced and digested with 10 mL of serum-free
DMEM containing 2 mg·mL−1 of collagenase Type II at 37 °C for 3 h. Then, tissue pieces were centrifuged at 2 000 r·min−1 for 10 min. After filtration using a stainless cell and centrifugation again at 2 000 r·min−1 for 10 min, the precipitations were incubated in DMEM with 15% FBS at 37 °C in 5% CO2 humidified atmosphere. After overnight culture, the adherent cells (FLS) were further cultured, non-adherent cells were removed, and the medium was changed after 24 h and every two days thereafter until confluent [14]. The subsequent experiments were performed using the cells within passages 3–6. The MH7A cells (cell line of human FLS) were obtained from Guangzhou Jenniobio Biotechnology Co., Ltd. (Guangzhou, China) and cultured with DMEM medium containing 10% FBS at 37 °C in 5% CO2 humidified atmosphere. Cell viability assay The cell viability was investigated by using MTT assay. Primary culture RA-FLS and MH7A cells were seeded in 96-well plates at a density of 1 × 104 cells/mL. After treated with various concentrations of Tet (0, 0.03, 0.1, 0.3, 1, 3, and 10 μmol·L−1) for 20 and 44 h, 20 μL of MTT (5 mg·mL−1) was added to each well and incubated for an additional 4 h. Subsequently, the supernatants were removed, and the formazone crystals were dissolved using 150 μL of DMSO. The absorbance at 570 nm was measured using a microplate reader (Thermo, Waltham, MA, USA). Cell proliferation assay The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in 96-well plates. After treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h, the proliferation of the cells was detected using a Cell-Light EdU Apollo 567 DNA kit (Ribibio, Guangzhou, China) according to the manufacturer’s instruction. Then, the images were obtained by using Olympus IX51 fluorescence microscopy. The proliferation rate was calculated by normalizing the number of EdU-positive cells to the Hoechst 33342 stained cells in ten random fields. Cell migration assay The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in 96-well plates. When 90% confluence was reached, one parallel “wound” in each well was created in the center of each well by scratching with a sterile P200 pipette tip. Serum-free medium was used to remove the debris. Then, the cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1), SB203580 (10 μmol·L−1), U0126 (10 μmol·L−1), SP600125 (10 μmol·L−1), Triciribine (10 μmol·L−1), LY294002 (10 μmol·L−1) and PDTC (10 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h. The wound area was photographed by using an Olympus IX51 light microscope at 0 h and 24 h. The extent of wound closure was presented as the percentage of the original scratch area. The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in the upper chambers of duplicate filters and treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the
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presence or absence of IL-1β (10 ng·mL−1) for 10 h. The non-migrating cells were removed from the upper surface by cotton swabs, and the cells migrated to the bottom were stained with crystal violet, photographed by using an Olympus IX51 light microscope, and counted in ten representative microscopic fields. Cell invasion assay The cell invasion assay was performed by using Matrigel invasion chambers (Milllpore, Billerica, USA)) according to the manufacturer’s instructions. The upper chambers were freshly coated with Matrigel, and the medium was added to the lower chamber. The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in the upper chambers of duplicate filters, and treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h. The cells on the top membrane surface were removed with cotton swabs, and those penetrated to the bottom were stained with crystal violet. Then, the images were obtained by using an Olympus IX51 fluorescence microscope. Gelatin zymography assay The activity of MMP-2/9 was detected by using the gelatin zymography assay, which was performed as described previously [15]. The MH7A cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 48 h in the presence or absence of IL-1β (10 ng·mL −1 ). The supernatants were collected and separated by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) co-polymerized with 1% gelatin. The gels were rinsed twice, incubated at 37 °C for 24 h in Tris-HCL buffer, stained with 0.25% Coomassie blue R250, and destained in a solution (10% methanol, 10% acetic acid), and the photographed by using an Olympus IX51 light microscope. Western blotting analysis The MH7A cells (1 × 104 cells/mL), seeded in 6-well plate, were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h, and stimulated with IL-1β (10 ng·mL−1) for different intervals. Afterwards, the cells were washed and lysed. The proteins were then centrifuged at 15, 000 r·min−1 for 5 min, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 10% non-fat milk in PBS- Tween (PBST) for 2 h, and incubated with relative primary antibodies. After being rinsed, the membranes were hybridized with secondary antibodies conjugated to horseradish peroxidase for 1 h. The protein bands of interest were visualized with ECL reagent. Statistical analysis The data were presented as means ± SEM. Statistical differences were assessed by one-way analysis of variance (ANOVA), followed by a post hoc Tukey’s test. P values less than 0.05 (P < 0.05) were considered statistically significant.
Results Effects of Tet on activity and proliferation of primary culture RA-FLS and MH7A cells To test the potential cytotoxic effects of Tet on primary
culture RA-FLS and MH7A cells, MTT assay was adopted. Our results showed that Tet-induced cytotoxicity in both FLS and MH7A cells for 24 h was negligible at 3 μmol·L−1 (Fig. 1A). Then, EdU-Click technology, an accurate approach for proliferation detection of various cells, was used to examine the effects of Tet on the proliferation of FLS. As shown in Fig. 1B, Tet only showed slight effect on the proliferation of the cells at 0.1–1 μmol·L−1. Effects of Tet on migration of primary culture RA-FLS and MH7A cells The wound healing and transwell assays are two classical methods for testing cell migration. We found that primary culture RA-FLS and MH7A cells in control group obviously migrated, and Tet (0.3, 1 μmol·L−1) treatments significantly inhibited their migration (Fig. 2). Effects of Tet on invasion of primary culture RA-FLS and MH7A cells RA-FLS have been proven to possess a property of excessive invasion as tumor cells [4-5]. In the present study, we detected the invasion of FLS by using Matrigel invasion chambers. Our results showed that invasion of both primary culture RA-FLS and MH7A cells in control group significantly increased, and Tet (0.3 and 1 μmol·L−1) treatments inhibited the invasion (Fig. 3). The data mentioned above manifested that Tet (0.3 and 1 μmol·L−1) could obviously reduce the migration and invasion of RA-FLS. Effects of Tet on activity and expression of MMP-2/9 in MH7A cells Cell migration and invasion are closely related to matrix degradation, a process mainly dependent on the activities of degradation enzymes such as MMPs [4]. In this experiment, IL-1β stimulation led to a significantly increased expression of MMP-2/9 in MH7A cells. Tet (0.3 and 1 μmol·L−1) treatments dramatically decreased the expression (Fig. 4A). In addition, the results from gelatin zymography assay showed that Tet (0.3 and 1 μmol·L−1) obviously reduced the activity of MMP-2/9 (Fig. 4B). Effects of Tet on the expression of F-actin and activation of FAK in MH7A cells Cell migration begins with protrusion of the plasma membrane at the leading edge, which is driven by polymerization of actin filaments and stabilized by formation of focal adhesions [10, 16]. For detection of cytoskeletal filamentous actin F-actin, the MH7A cells were incubated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h, and the expression of F-actin was detected by Western blotting assay. As shown in Fig. 5A, the F-actin expression was obviously down-regulated by Tet (0.3 and 1 μmol·L−1) treatments. FAK is one of the most important focal adhesion proteins located at the focal contacts [10, 16]. In the present study, the activation of FAK was detected. Our results showed that IL-1β stimulation markedly promoted the phosphorylation of FAK in MH7A cells, which was largely reduced by Tet (0.3 and 1 μmol·L−1) treatments (Fig. 5B).
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Fig. 1 Effects of tetrandrine (Tet) on the viability and proliferation of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and the MH7A cells. (A) Viability of cells was determined by using MTT assay. (B) Proliferation of cells induced by IL-1β was determined by using EdU assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal
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Fig. 2 Effects of tetrandrine (Tet) on migration of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and MH7A cells induced by IL-1β. (A) Migration of cells was determined by using wounding healing assay. (B) Migration of cells was determined by using transwell assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Effects of Tet on the expressions of Rac1, Cdc42, and RhoA in MH7A cells The data in mountain have demonstrated that proteins in Rho family play important roles in cell migration [17]. They mediate the polymerization of actin and induce the formation and extension of pseudopodia. Among them,
Rac1, Cdc42 and RhoA have attracted much attention [18]. After stimulated with IL-1β (10 ng·mL−1), the MH7A cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h. Our results showed that Tet (0.3 and 1 μmol·L−1) could reduce the expressions of Rac1, Cdc42 and RhoA in the MH7A cells (Fig. 6).
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Fig. 3 Effects of tetrandrine (Tet) on the invasion of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and MH7A cells induced by IL-1β. Cell invasion assay was performed by using Matrigel invasion chambers. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Fig. 4 Effects of tetrandrine (Tet) on the activities and expressions of MMP-2/9 induced by IL-1β in MH7A cells. (A) Activities of MMP-2/9 were determined by using gelatin zymography assay. (B) Expressions of MMP-2/9 were determined by using western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
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Fig. 5 Effects of tetrandrine (Tet) on the activation of FAK and the expression of F-actin in MH7A cells induced by IL-1β. (A) Expression of F-actin was determined by using Western blotting assay. (B) Expressions of FAK and p-FAK were determined by using western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; * P < 0.05, **P < 0.01 vs control
Fig. 6 Effects of tetrandrine (Tet) on the expressions of Rac1, Cdc42 and RhoA in MH7A cells induced by IL-1β. Expressions of Rac1, Cdc42 and RhoA were determined by using Western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Involvement of signaling pathways in migration of MH7A cells Although accumulative data show that PI3K/Akt, NF-κB and MAPK signaling pathways are involved in the migration process of various cells, their roles in the migration of RA-FLS remain obscure [19-21]. In the present study, the specific inhibitors (SB203580, an inhibitor of p38 MAPK; U0126, an inhibitor of ERK; SP600125, an inhibitor of JNK; Triciribine, an inhibitor of Akt;
LY294002, an inhibitor of PI3K; and PDTC, an inhibitor of NF-κB) were used in wound healing assay to identify which signaling pathway(s) play a key role in the migration of MH7A cells. Our results presented in Fig. 7A showed that LY294002, Triciribine, and SP600125 (10 μmol·L−1) could inhibit the migration of the MH7A cells, and SP600125 exhibited most potent inhibition, suggesting that PI3K/Akt and JNK signaling pathways might be more important for the migration of RA-FLS.
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Fig. 7 Effects of tetrandrine (Tet) on activation of PI3K/Akt and JNK signaling pathways induced by IL-1β in MH7A cells. (A) Effects of specific signaling inhibitors on migration of MH7A cells were determined by using wounding healing assay. (B) Expressions of Akt, p-Akt, JNK and p-JNK were determined by using Western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
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Effects of Tet on the activation of PI3K/Akt and JNK in MH7A cells After being activated by various cytokines and factors including Rho/Rho-kinase, PI3K/Akt and JNK can induce the expressions of many proteins such as MMP-9 and F-actin, and finally mediate cell migration [19-21]. In the present study, the effects of Tet on the activation of Akt and JNK in MH7A cells were observed. As shown in Fig. 7B, IL-1β stimulation resulted in marked phosphorylations of JNK and Akt in MH7A cells, which was significantly down-regulated by Tet (0.3 and 1 μmol·L−1) treatments.
Discussion RA is a chronic autoimmune disease characterized by abnormal synovial hyperplasia and progressive destruction of cartilage and bone [4]. Various cell types, such as T cells, B cells, macrophages, osteoclasts, and FLS are implicated in the destructive progress of joints. FLS, mainly present in synovium, exhibit the characteristics of tumor cells and play a critical role in the development of pannus by migrating into cartilage and bone [5]. More importantly, a recent in vivo study using severe combined immune-deficient (SCID) mouse model shows that long-distance transmigration of activated RA-FLS may mediate, at least in part, the destructive arthritis spreading between joints [22] These studies indicate that modulation of migration and invasion of activated-FLS may be a novel therapeutic strategy for destructive progress of RA. In RA, the migration of FLS is mainly due to two factors: first, perceiving stimulation from inflammatory cytokines and soluble factors such as IL-1β, LPS, TNF-α and PDGF; and second, feeling ligands such as integrins at fixed positions within the synovial membranes [23-26]. As the micro-environment of synovium in RA is structured by lots of pro-inflammatory cytokines, IL-1β was selected as an inducer of FLS migration in this study. Our results from the present study showed that Tet could inhibit migration and invasion of both primary culture RA-FLS and MH7A cells, without marked effects on cell activity and proliferation. The migration and invasion of RA-FLS involve multiple steps such as degradation of extracellular matrix, formation and extension of cell pseudopodia, reconstruction of cytoskeleton, and formation of focal adhesion [4, 10, 16]. The intervention mechanism of Tet needs to be identified in the future. MMPs, a family of zinc-dependent endopeptidases, are the main proteases for invasion and degradation of basement membranes and extracellular matrix. They are initially expressed in an enzymatically inactive state due to the interaction of a cysteine residue of the pro-domain with the zinc ion of the catalytic site. MMP-2 (also known as gelatinase-A) and MMP-9 (also known as gelatinase-B), key members of MMPs family, are capable of cleaving gelatine, type I, IV, and V collagens, elastin and vitronectin, and provide condition for the cell migration and invasion [27-28]. In
RA, greatly higher levels of MMP-9 and MMP-2 in peripheral blood and synovial fluid than osteoarthritis are found [27]. In addition, MMP-9 knockout mice show reduced severity of antibody-induced arthritis [28]; MMP-9 siRNA has inhibitory effects on migration and invasion of RA-FLS and could decrease the production of IL-1β, IL-6, IL-8 and TNF-α, inactivate NF-κB, ERK and JNK and suppress RA-FLSmediated cartilage degradation [29]. All these results indicate that MMP-2/9 play important roles in migration and invasion of RA-FLS. In the present study, Tet (0.3 and 1 μmol·L−1) obviously reduced the activity and expression of MMP-2/9 in RA-FLS. Cytoskeleton is a structure composed of actin filaments, microtubules and intermediate filaments in the cytoplasm, and has functions of supporting and maintaining cell morphology and movement [9]. Actin consists the microfilament in eukaryotic cells. In FLS, the actin shows as fibers, and called as F-actin, which participates filopodia formation of cytoskeleton changes during cell migration [10-11]. In addition, focal adhesion acts as the attachment point of extracellular matrix and cytoskeleton, and guides direction for the cell migration. Focal adhesion kinase (FAK), a 125 kD non-receptor protein tyrosine kinase, is the key signaling nexus connecting integrins and the dynamic actin cytoskeleton. It can catalyze tyrosine phosphorylation of target proteins on the focal adhesion to coordinate cell migration and invasion [30]. Our results from the present study revealed that Tet inhibited the expression of F-actin and activation of FAK in MH7A cells. The Rho family of small GTPases forms a 20-member family within Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well-known Rho family members include RhoA, Cdc42, and Rac1 [19-20]. Rac1 regulates the formation of membrane protrusions (lamellipodia) at the leading edge of migrating cells, whereas RhoA regulates the assembly of actin stress fibers through its downstream effectors mDIA and ROCKs and limits the extent of lamellipodium [31]. Then, filopodia, formed after the forefront of actin polymerization of microfilament, assumes the needlepoint sample protrusions, mainly regulated by Cdc42 [32]. Increased activities of RhoA and ROCK are found in ex vivo FLS from RA patients, compared with that of osteoarthritis patients and healthy control, and inhibition of RhoA and ROCK activities would suppress the cytoskeletal reorganization and proliferation of RA-FLS [33]. In addition, Rac1 can inhibit RA-FLS proliferation and invasion to a similar extent as NSC23766 (a Rac1 inhibitor) [33-34]. Therefore, we investigated the influence of Tet on expression of Rho proteins, and our results showed that Tet significantly inhibited the expressions of RhoA, Rac1, and Cdc42 in MH7A cells. MAPK, NF-κB, and PI3K/Akt all have been reported to play important roles in the migration and invasion of various cells, but their participation in that of RA-FLS is
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still obscure [19-21]. Various pathological factors (pro-inflammatory cytokines, chemotactic factors) in RA can activate these signaling pathways by binding with corresponding receptors to regulate the expressions of downstream factors, control the actin cytoskeleton, and then mediate the migration and invasion of cells [35-37]. By using specific signaling inhibitors of MAPK, NF-κB and PI3K/Akt in the present study, we found that PI3K/Akt and JNK were deeply involved in the migration of MH7A cells, which was in accordance with the report that Rho proteins-mediated activation of PI3K/Akt and JNK could contribute to the behavior of RA-FLS [35-37]. Our further Western blotting resutls indicated that Tet could significantly reduce that activation of JNK and Akt in MH7A cells.
Conclusions In summary, Tet could markedly inhibit the migration and invasion of RA-FLS. The mechanisms may involve the down-regulation of Rac1, Cdc42, and RhoA expressions, inhibition of PI3K/Akt and JNK activation, and ultimate prevention of activation and/or expressions of MMP-9, F-actin, and FAK.
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Cite this article as: LV Qi, ZHU Xian-Yang, XIA Yu-Feng, DAI Yue, WEI Zhi-Feng. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42 and RhoA GTPases and activation of PI3K/Akt and JNK signaling pathways [J]. Chinese Journal of Natural Medicines, 2015, 13(11): 831-841
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Chinese Journal of Natural Medicines
Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways LV Qi, ZHU Xian-Yang △, XIA Yu-Feng, DAI Yue *, WEI Zhi-Feng * Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China Available online 20 Nov., 2015
[ABSTRACT] Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L−1) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. [KEY WORDS] Tetrandrine; Rheumatoid arthritis; Fibroblast-like synoviocytes; Migration; Invasion
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)11-0831-11
Introduction Rheumatoid arthritis (RA) is characterized by chronic
[Received on] 16-May-2015 [Research funding] This work was partially supported by the National Natural Science Foundation of China (No. 81373426) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. [*Corresponding author] Tel: +86-25-83271400, Fax: +86-2585301528, E-mail: [email protected] (WEI Zhi-Feng); yuedaicpu@ hotmail.com (DAI Yue) △ Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
synovitis, which causes synovial hyperplasia and ultimate joint damage [1-3]. Fibroblast-like synoviocytes (FLS), located at synovial intimal edge, are identified as key players in the pathophysiological process of RA [4]. There are reports indicating that RA-FLS share many similar properties with tumor cells: undergoing tumor-like proliferation, migration and invasion, as well as possessing increased resistance to apoptosis [5]. They can migrate to the unaffected joints, attach to cartilage and bone, and invade into the extracellular matrix [6]. All these findings suggest that FLS are important effectors for spreading arthritis destruction to distant joints. Migration and invasion are complex processes that need dynamic interactions between cells and the surrounding matrix, and coordinated programs executed by multiple pathways that integrate signals from the extracellular environment and remodeling the actin cytoskeleton [7]. It is
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well established that pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α which are deeply involved in the pathogenesis of RA, constitute key mediators of RA-FLS migration and invasion [8]. Cytokines, as activators of FLS, lead to activation of Rho GTPases and signaling pathways (including NF-κB, MAPK and PI3K/Akt) by binding with specific acceptors, subsequently up-regulating the expressions of effector proteins such as degrading enzyme MMP-9 and F-actin, and then mediating the migration and invasion of cells [9-10]. Targeted modulation of the signaling pathways that control the expressions of migration- and invasion-related proteins may be helpful for the intervention of RA. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, has been clinically used for the management of RA-related diseases in China for a long time. By oral administration, Tet could ameliorate adjuvant-induced arthritis in rats, a well-established animal model of human RA [11]. However, its mechanism remains to be elucidated. Recent evidence indicates that Tet could inhibit migration and invasion of various kinds of tumor cells such as human prostate cancer DU145 and PC-3 cells and lung cancer A549 cells [12-13]. Those findings attracted us to explore the anti-RA mechanism of Tet based on migration and invasion of FLS in the present study.
Materials and Methods Chemicals and reagents Tet (purity > 95%) was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). IL-1β was purchased from PeproTech Int. (Rocky Hill, CT, USA). Dulbecco’s modified eagle medium (DMEM) was purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Wisent Corporation (Nanjing, China). 24-well transwell inserts were purchased from Milllpore (Massachusetts, Billerica, USA). Cell-Light EdU Apollo 567 DNA kit was purchased from RiboBio Co. Ltd. (Guangzhou, China). Monoclonal antibodies against GAPDH, MMP- 2/9, p38, p-p38, JNK, p-JNK, ERK, p-ERK, Akt, p-Akt, F-actin, FAK, p-FAK, Rac1, Cdc42, and RhoA were purchased from Bioworld (Minneapolis, MN, USA). SB203580 (a specific inhibitor of p38 MAPK), U0126 (a specific inhibitor of ERK), SP600125 (a specific inhibitor of JNK), LY294002 (a specific inhibitor of PI3K), and Triciribine (a specific inhibitor of Akt) were purchased from KangChen Bio-tech (Shanghai, China). The other chemicals and reagents were of analytical grade and obtained from commercial sources. Cell cultures The synovial tissues of RA patients were collected during knee joint arthroscopy according to the protocol approved by Institutional Ethics Review Committee of the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. The tissues were minced and digested with 10 mL of serum-free
DMEM containing 2 mg·mL−1 of collagenase Type II at 37 °C for 3 h. Then, tissue pieces were centrifuged at 2 000 r·min−1 for 10 min. After filtration using a stainless cell and centrifugation again at 2 000 r·min−1 for 10 min, the precipitations were incubated in DMEM with 15% FBS at 37 °C in 5% CO2 humidified atmosphere. After overnight culture, the adherent cells (FLS) were further cultured, non-adherent cells were removed, and the medium was changed after 24 h and every two days thereafter until confluent [14]. The subsequent experiments were performed using the cells within passages 3–6. The MH7A cells (cell line of human FLS) were obtained from Guangzhou Jenniobio Biotechnology Co., Ltd. (Guangzhou, China) and cultured with DMEM medium containing 10% FBS at 37 °C in 5% CO2 humidified atmosphere. Cell viability assay The cell viability was investigated by using MTT assay. Primary culture RA-FLS and MH7A cells were seeded in 96-well plates at a density of 1 × 104 cells/mL. After treated with various concentrations of Tet (0, 0.03, 0.1, 0.3, 1, 3, and 10 μmol·L−1) for 20 and 44 h, 20 μL of MTT (5 mg·mL−1) was added to each well and incubated for an additional 4 h. Subsequently, the supernatants were removed, and the formazone crystals were dissolved using 150 μL of DMSO. The absorbance at 570 nm was measured using a microplate reader (Thermo, Waltham, MA, USA). Cell proliferation assay The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in 96-well plates. After treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h, the proliferation of the cells was detected using a Cell-Light EdU Apollo 567 DNA kit (Ribibio, Guangzhou, China) according to the manufacturer’s instruction. Then, the images were obtained by using Olympus IX51 fluorescence microscopy. The proliferation rate was calculated by normalizing the number of EdU-positive cells to the Hoechst 33342 stained cells in ten random fields. Cell migration assay The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in 96-well plates. When 90% confluence was reached, one parallel “wound” in each well was created in the center of each well by scratching with a sterile P200 pipette tip. Serum-free medium was used to remove the debris. Then, the cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1), SB203580 (10 μmol·L−1), U0126 (10 μmol·L−1), SP600125 (10 μmol·L−1), Triciribine (10 μmol·L−1), LY294002 (10 μmol·L−1) and PDTC (10 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h. The wound area was photographed by using an Olympus IX51 light microscope at 0 h and 24 h. The extent of wound closure was presented as the percentage of the original scratch area. The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in the upper chambers of duplicate filters and treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the
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presence or absence of IL-1β (10 ng·mL−1) for 10 h. The non-migrating cells were removed from the upper surface by cotton swabs, and the cells migrated to the bottom were stained with crystal violet, photographed by using an Olympus IX51 light microscope, and counted in ten representative microscopic fields. Cell invasion assay The cell invasion assay was performed by using Matrigel invasion chambers (Milllpore, Billerica, USA)) according to the manufacturer’s instructions. The upper chambers were freshly coated with Matrigel, and the medium was added to the lower chamber. The primary culture RA-FLS and MH7A cells (1 × 104 cells/mL) were seeded in the upper chambers of duplicate filters, and treated with Tet (0.1, 0.3, and 1 μmol·L−1) in the presence or absence of IL-1β (10 ng·mL−1) for 24 h. The cells on the top membrane surface were removed with cotton swabs, and those penetrated to the bottom were stained with crystal violet. Then, the images were obtained by using an Olympus IX51 fluorescence microscope. Gelatin zymography assay The activity of MMP-2/9 was detected by using the gelatin zymography assay, which was performed as described previously [15]. The MH7A cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 48 h in the presence or absence of IL-1β (10 ng·mL −1 ). The supernatants were collected and separated by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) co-polymerized with 1% gelatin. The gels were rinsed twice, incubated at 37 °C for 24 h in Tris-HCL buffer, stained with 0.25% Coomassie blue R250, and destained in a solution (10% methanol, 10% acetic acid), and the photographed by using an Olympus IX51 light microscope. Western blotting analysis The MH7A cells (1 × 104 cells/mL), seeded in 6-well plate, were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h, and stimulated with IL-1β (10 ng·mL−1) for different intervals. Afterwards, the cells were washed and lysed. The proteins were then centrifuged at 15, 000 r·min−1 for 5 min, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 10% non-fat milk in PBS- Tween (PBST) for 2 h, and incubated with relative primary antibodies. After being rinsed, the membranes were hybridized with secondary antibodies conjugated to horseradish peroxidase for 1 h. The protein bands of interest were visualized with ECL reagent. Statistical analysis The data were presented as means ± SEM. Statistical differences were assessed by one-way analysis of variance (ANOVA), followed by a post hoc Tukey’s test. P values less than 0.05 (P < 0.05) were considered statistically significant.
Results Effects of Tet on activity and proliferation of primary culture RA-FLS and MH7A cells To test the potential cytotoxic effects of Tet on primary
culture RA-FLS and MH7A cells, MTT assay was adopted. Our results showed that Tet-induced cytotoxicity in both FLS and MH7A cells for 24 h was negligible at 3 μmol·L−1 (Fig. 1A). Then, EdU-Click technology, an accurate approach for proliferation detection of various cells, was used to examine the effects of Tet on the proliferation of FLS. As shown in Fig. 1B, Tet only showed slight effect on the proliferation of the cells at 0.1–1 μmol·L−1. Effects of Tet on migration of primary culture RA-FLS and MH7A cells The wound healing and transwell assays are two classical methods for testing cell migration. We found that primary culture RA-FLS and MH7A cells in control group obviously migrated, and Tet (0.3, 1 μmol·L−1) treatments significantly inhibited their migration (Fig. 2). Effects of Tet on invasion of primary culture RA-FLS and MH7A cells RA-FLS have been proven to possess a property of excessive invasion as tumor cells [4-5]. In the present study, we detected the invasion of FLS by using Matrigel invasion chambers. Our results showed that invasion of both primary culture RA-FLS and MH7A cells in control group significantly increased, and Tet (0.3 and 1 μmol·L−1) treatments inhibited the invasion (Fig. 3). The data mentioned above manifested that Tet (0.3 and 1 μmol·L−1) could obviously reduce the migration and invasion of RA-FLS. Effects of Tet on activity and expression of MMP-2/9 in MH7A cells Cell migration and invasion are closely related to matrix degradation, a process mainly dependent on the activities of degradation enzymes such as MMPs [4]. In this experiment, IL-1β stimulation led to a significantly increased expression of MMP-2/9 in MH7A cells. Tet (0.3 and 1 μmol·L−1) treatments dramatically decreased the expression (Fig. 4A). In addition, the results from gelatin zymography assay showed that Tet (0.3 and 1 μmol·L−1) obviously reduced the activity of MMP-2/9 (Fig. 4B). Effects of Tet on the expression of F-actin and activation of FAK in MH7A cells Cell migration begins with protrusion of the plasma membrane at the leading edge, which is driven by polymerization of actin filaments and stabilized by formation of focal adhesions [10, 16]. For detection of cytoskeletal filamentous actin F-actin, the MH7A cells were incubated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h, and the expression of F-actin was detected by Western blotting assay. As shown in Fig. 5A, the F-actin expression was obviously down-regulated by Tet (0.3 and 1 μmol·L−1) treatments. FAK is one of the most important focal adhesion proteins located at the focal contacts [10, 16]. In the present study, the activation of FAK was detected. Our results showed that IL-1β stimulation markedly promoted the phosphorylation of FAK in MH7A cells, which was largely reduced by Tet (0.3 and 1 μmol·L−1) treatments (Fig. 5B).
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Fig. 1 Effects of tetrandrine (Tet) on the viability and proliferation of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and the MH7A cells. (A) Viability of cells was determined by using MTT assay. (B) Proliferation of cells induced by IL-1β was determined by using EdU assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal
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Fig. 2 Effects of tetrandrine (Tet) on migration of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and MH7A cells induced by IL-1β. (A) Migration of cells was determined by using wounding healing assay. (B) Migration of cells was determined by using transwell assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Effects of Tet on the expressions of Rac1, Cdc42, and RhoA in MH7A cells The data in mountain have demonstrated that proteins in Rho family play important roles in cell migration [17]. They mediate the polymerization of actin and induce the formation and extension of pseudopodia. Among them,
Rac1, Cdc42 and RhoA have attracted much attention [18]. After stimulated with IL-1β (10 ng·mL−1), the MH7A cells were treated with Tet (0.1, 0.3, and 1 μmol·L−1) for 24 h. Our results showed that Tet (0.3 and 1 μmol·L−1) could reduce the expressions of Rac1, Cdc42 and RhoA in the MH7A cells (Fig. 6).
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Fig. 3 Effects of tetrandrine (Tet) on the invasion of primary fibroblast-like synoviocytes from patients of rheumatoid arthritis (RA-FLS) and MH7A cells induced by IL-1β. Cell invasion assay was performed by using Matrigel invasion chambers. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Fig. 4 Effects of tetrandrine (Tet) on the activities and expressions of MMP-2/9 induced by IL-1β in MH7A cells. (A) Activities of MMP-2/9 were determined by using gelatin zymography assay. (B) Expressions of MMP-2/9 were determined by using western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
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Fig. 5 Effects of tetrandrine (Tet) on the activation of FAK and the expression of F-actin in MH7A cells induced by IL-1β. (A) Expression of F-actin was determined by using Western blotting assay. (B) Expressions of FAK and p-FAK were determined by using western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; * P < 0.05, **P < 0.01 vs control
Fig. 6 Effects of tetrandrine (Tet) on the expressions of Rac1, Cdc42 and RhoA in MH7A cells induced by IL-1β. Expressions of Rac1, Cdc42 and RhoA were determined by using Western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
Involvement of signaling pathways in migration of MH7A cells Although accumulative data show that PI3K/Akt, NF-κB and MAPK signaling pathways are involved in the migration process of various cells, their roles in the migration of RA-FLS remain obscure [19-21]. In the present study, the specific inhibitors (SB203580, an inhibitor of p38 MAPK; U0126, an inhibitor of ERK; SP600125, an inhibitor of JNK; Triciribine, an inhibitor of Akt;
LY294002, an inhibitor of PI3K; and PDTC, an inhibitor of NF-κB) were used in wound healing assay to identify which signaling pathway(s) play a key role in the migration of MH7A cells. Our results presented in Fig. 7A showed that LY294002, Triciribine, and SP600125 (10 μmol·L−1) could inhibit the migration of the MH7A cells, and SP600125 exhibited most potent inhibition, suggesting that PI3K/Akt and JNK signaling pathways might be more important for the migration of RA-FLS.
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Fig. 7 Effects of tetrandrine (Tet) on activation of PI3K/Akt and JNK signaling pathways induced by IL-1β in MH7A cells. (A) Effects of specific signaling inhibitors on migration of MH7A cells were determined by using wounding healing assay. (B) Expressions of Akt, p-Akt, JNK and p-JNK were determined by using Western blotting assay. The data are expressed as means ± SEM of three independent experiments. ##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs control
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Effects of Tet on the activation of PI3K/Akt and JNK in MH7A cells After being activated by various cytokines and factors including Rho/Rho-kinase, PI3K/Akt and JNK can induce the expressions of many proteins such as MMP-9 and F-actin, and finally mediate cell migration [19-21]. In the present study, the effects of Tet on the activation of Akt and JNK in MH7A cells were observed. As shown in Fig. 7B, IL-1β stimulation resulted in marked phosphorylations of JNK and Akt in MH7A cells, which was significantly down-regulated by Tet (0.3 and 1 μmol·L−1) treatments.
Discussion RA is a chronic autoimmune disease characterized by abnormal synovial hyperplasia and progressive destruction of cartilage and bone [4]. Various cell types, such as T cells, B cells, macrophages, osteoclasts, and FLS are implicated in the destructive progress of joints. FLS, mainly present in synovium, exhibit the characteristics of tumor cells and play a critical role in the development of pannus by migrating into cartilage and bone [5]. More importantly, a recent in vivo study using severe combined immune-deficient (SCID) mouse model shows that long-distance transmigration of activated RA-FLS may mediate, at least in part, the destructive arthritis spreading between joints [22] These studies indicate that modulation of migration and invasion of activated-FLS may be a novel therapeutic strategy for destructive progress of RA. In RA, the migration of FLS is mainly due to two factors: first, perceiving stimulation from inflammatory cytokines and soluble factors such as IL-1β, LPS, TNF-α and PDGF; and second, feeling ligands such as integrins at fixed positions within the synovial membranes [23-26]. As the micro-environment of synovium in RA is structured by lots of pro-inflammatory cytokines, IL-1β was selected as an inducer of FLS migration in this study. Our results from the present study showed that Tet could inhibit migration and invasion of both primary culture RA-FLS and MH7A cells, without marked effects on cell activity and proliferation. The migration and invasion of RA-FLS involve multiple steps such as degradation of extracellular matrix, formation and extension of cell pseudopodia, reconstruction of cytoskeleton, and formation of focal adhesion [4, 10, 16]. The intervention mechanism of Tet needs to be identified in the future. MMPs, a family of zinc-dependent endopeptidases, are the main proteases for invasion and degradation of basement membranes and extracellular matrix. They are initially expressed in an enzymatically inactive state due to the interaction of a cysteine residue of the pro-domain with the zinc ion of the catalytic site. MMP-2 (also known as gelatinase-A) and MMP-9 (also known as gelatinase-B), key members of MMPs family, are capable of cleaving gelatine, type I, IV, and V collagens, elastin and vitronectin, and provide condition for the cell migration and invasion [27-28]. In
RA, greatly higher levels of MMP-9 and MMP-2 in peripheral blood and synovial fluid than osteoarthritis are found [27]. In addition, MMP-9 knockout mice show reduced severity of antibody-induced arthritis [28]; MMP-9 siRNA has inhibitory effects on migration and invasion of RA-FLS and could decrease the production of IL-1β, IL-6, IL-8 and TNF-α, inactivate NF-κB, ERK and JNK and suppress RA-FLSmediated cartilage degradation [29]. All these results indicate that MMP-2/9 play important roles in migration and invasion of RA-FLS. In the present study, Tet (0.3 and 1 μmol·L−1) obviously reduced the activity and expression of MMP-2/9 in RA-FLS. Cytoskeleton is a structure composed of actin filaments, microtubules and intermediate filaments in the cytoplasm, and has functions of supporting and maintaining cell morphology and movement [9]. Actin consists the microfilament in eukaryotic cells. In FLS, the actin shows as fibers, and called as F-actin, which participates filopodia formation of cytoskeleton changes during cell migration [10-11]. In addition, focal adhesion acts as the attachment point of extracellular matrix and cytoskeleton, and guides direction for the cell migration. Focal adhesion kinase (FAK), a 125 kD non-receptor protein tyrosine kinase, is the key signaling nexus connecting integrins and the dynamic actin cytoskeleton. It can catalyze tyrosine phosphorylation of target proteins on the focal adhesion to coordinate cell migration and invasion [30]. Our results from the present study revealed that Tet inhibited the expression of F-actin and activation of FAK in MH7A cells. The Rho family of small GTPases forms a 20-member family within Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well-known Rho family members include RhoA, Cdc42, and Rac1 [19-20]. Rac1 regulates the formation of membrane protrusions (lamellipodia) at the leading edge of migrating cells, whereas RhoA regulates the assembly of actin stress fibers through its downstream effectors mDIA and ROCKs and limits the extent of lamellipodium [31]. Then, filopodia, formed after the forefront of actin polymerization of microfilament, assumes the needlepoint sample protrusions, mainly regulated by Cdc42 [32]. Increased activities of RhoA and ROCK are found in ex vivo FLS from RA patients, compared with that of osteoarthritis patients and healthy control, and inhibition of RhoA and ROCK activities would suppress the cytoskeletal reorganization and proliferation of RA-FLS [33]. In addition, Rac1 can inhibit RA-FLS proliferation and invasion to a similar extent as NSC23766 (a Rac1 inhibitor) [33-34]. Therefore, we investigated the influence of Tet on expression of Rho proteins, and our results showed that Tet significantly inhibited the expressions of RhoA, Rac1, and Cdc42 in MH7A cells. MAPK, NF-κB, and PI3K/Akt all have been reported to play important roles in the migration and invasion of various cells, but their participation in that of RA-FLS is
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still obscure [19-21]. Various pathological factors (pro-inflammatory cytokines, chemotactic factors) in RA can activate these signaling pathways by binding with corresponding receptors to regulate the expressions of downstream factors, control the actin cytoskeleton, and then mediate the migration and invasion of cells [35-37]. By using specific signaling inhibitors of MAPK, NF-κB and PI3K/Akt in the present study, we found that PI3K/Akt and JNK were deeply involved in the migration of MH7A cells, which was in accordance with the report that Rho proteins-mediated activation of PI3K/Akt and JNK could contribute to the behavior of RA-FLS [35-37]. Our further Western blotting resutls indicated that Tet could significantly reduce that activation of JNK and Akt in MH7A cells.
Conclusions In summary, Tet could markedly inhibit the migration and invasion of RA-FLS. The mechanisms may involve the down-regulation of Rac1, Cdc42, and RhoA expressions, inhibition of PI3K/Akt and JNK activation, and ultimate prevention of activation and/or expressions of MMP-9, F-actin, and FAK.
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Cite this article as: LV Qi, ZHU Xian-Yang, XIA Yu-Feng, DAI Yue, WEI Zhi-Feng. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42 and RhoA GTPases and activation of PI3K/Akt and JNK signaling pathways [J]. Chinese Journal of Natural Medicines, 2015, 13(11): 831-841
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