Inflammation ( # 2015) DOI: 10.1007/s10753-015-0205-2
KPNA2 Contributes to the Inflammatory Processes in Synovial Tissue of Patients with Rheumatoid Arthritis and SW982 Cells Zhongbing Liu,1 Dongmei Zhang,1,2 Chi Sun,1 Ran Tao,1 Xinbao Xu,1 Libin Xu,1 Hongbing Cheng,3 Min Xiao,4 and Youhua Wang1,5
Abstract—Karyopherin-α2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. We investigated the function and possible mechanisms of KPNA2 involved in rheumatoid arthritis (RA). Western blotting and immunohistochemistry showed the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Double immunofluorescent staining indicated that KPNA2 co-localized with T cells, macrophage-like synoviocytes, fibroblast-like synoviocytes, and neutrophils in synovial tissue of RA patients. Moreover, the expression of KPNA2 in SW982 cells was increased in a time-dependent manner in response to TNFα stimulation. Both Western blotting and immunofluorescent staining assay revealed the co-localization of KPNA2 and P65 and their translocation from cytoplasma in TNFα-treated SW982 cells. Furthermore, knocking down the expression of KPNA2 by siRNA inhibited TNFα-induced expression of IL-6, MMP-1, and MMP-13 and, more importantly, decreased the P65 phosphorylation in SW982 cells. We therefore suggested that KPNA2 may play a key role in the inflammation process of RA via NF-κB P65 signal transduction pathway. KEY WORDS: karyopherin-α2 (KPNA2); rheumatoid arthritis (RA); SW982; tumor necrosis factor-α (TNFα); P65.
INTRODUCTION Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease which is characterized by infiltration of inflammatory cell and resulting in synovial hyperplasia and joint destruction [1, 2]. The pathogenesis of proliferation has still not been clarified, but the characteristics of Zhongbing Liu and Dongmei Zhang contributed equally to this work. 1
Department of Orthopaedics, Affiliated Hospital of Nantong University, and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001Jiangsu Province, China 2 Department of Pathogen Biology, Medical College, Nantong University, Nantong, 226001, China 3 Department of Orthopaedics, Traditional Chinese Medical Hospital of Nantong City, Nantong, 226001, China 4 National Glycoengineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China 5 To whom correspondence should be addressed at Department of Orthopaedics, Affiliated Hospital of Nantong University, and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001Jiangsu Province, China. E-mail: [email protected]
RA synoviocytes are similar to those of transformed cells [3]. NF-κB, one of the major transcription factor implicated in joint inflammation, is essential for the production of cytokines and proteases by RA fibroblast-like synoviocytes (FLSs), and it mediates the resistance of RA FLS to apoptosis [4–6]. In humans, the synovial cell is a major source of inflammatory cytokines such as IL-1, IL-6, and TNFα, all of which play a chief role during the development of RA [7]. Because of these cytokines, NFκB can be activated and further cause the expression of various inflammatory mediators. Karyopherin-α2 (KPNA2), a member of the importin family, is a kind of nuclear transport proteins [8]. One of the last major steps of the most intracellular signaling pathways is nucleocytoplasmic transport [9]. Some proteins such as transcription factors in the cytoplasm enter the nucleus and modulate the expression of the effect genes or other signaling molecules. Nucleocytoplasmic transport of the molecules within the cytoplasm occurs through large nuclear pore complexes in the nuclear membrane [10]. Preliminary studies showed that KPNA2 was associated
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang with cell proliferation and tumor formation, and people paid more and more attention to its importance [11]. However, the expression of KPNA2 and its biological function in RA remains to be elucidated. The human synovial cell line, SW982, is a useful tool to investigate the expression of inflammatory cytokines or MMPs in response to TNFα [12, 13]. In this study, we first investigated the expression of KPNA2 in synovial tissue of patients with RA and SW982 cells and explored the mechanism involved in the RA pathogenesis.
MATERIALS AND METHODS Patients and RASFs There were five females and five males in our cohort of RA patients. Samples were obtained from patients at the time of total knee replacement surgery. All patients were diagnosed according to the standards defined by the American College of Rheumatology in 1987. The disease activity was calculated by using the disease activity score (DAS), and, for the current study only, patients with a DAS higher than 3.2 were included [14, 15]. The synovial tissue from ten healthy individuals was isolated during arthroscopic procedures performed by orthopedic surgeons as control. Age in the control group was 45±5 years and for the RA patients was 46±6 years. The study was approved by the institutional medical ethics committee of the Affiliated Hospital of Nantong University and informed consent was obtained from all patients prior to surgery. Cell Culture and Stimulation Synovial tissue was digested for 2 h with 0.25 % (weight/volume) collagenase and then suspended in RPMI 1640 medium with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in 5 % CO2 for several days, after which nonadherent cells were removed. RASFs from passages 3 to 7 were used for our experiment. The human synovial cell line SW982 was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in humidified air with 5 % CO2. The medium was changed every 2–3 days, and cell passage was carried out every 5–6 days. For in vitro experiments in SW982 cells, cells were incubated with serum-free DMEM for 24 h and then stimulated with
TNFα (Human, Sigma Chemical Co., St. Louis, MO, USA) at the indicated concentration and time. The cells were harvested after treatment and used for further analysis. Western Blot Analysis The synovial tissue samples of RA patients and healthy individuals were homogenized in lysis buffer (1 % NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l ethylene diamine tetraacetic acid (EDTA), 1 % sodium dodecyl sulfate (SDS), 1 % sodium deoxycholate, 1 % Triton X-100, 1 mmol/l phenylmethyl sulfonyl fluoride (PMSF), 10 lg/ml aprotinin, and 1 lg/ml leupeptin) and then centrifuged at 12,000 and 4 rpm for 20 min to collect the supernatant. Cell cultures for immunoblot were lysed in buffer (10 mM tris, pH 8.0,150 mM NaCl, 1 % nonidet P-40, 0.5 % deoxycholate, 0.1 % SDS and protease inhibitor mixture) followed by centrifugation to discard cell debris. To prepare cytoplasmic and nuclear extracts of cells, we homogenized frozen samples in the above buffer, then centrifuged them at 14,000 rpm for 1 min at 4 °C, and finally stored cytoplasmic extracts at −80 °C at once. We resuspended nuclei in a low-salt buffer and agitated. Samples were centrifuged at 14,000 rpm for 5 min at 4 °C, and nuclear extracts were stored at −80 °C immediately. After determining the protein concentration with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene diflouride filter (PVDF) membranes (Millipore, Bed ford, MA, USA). The membranes were blocked with 5 % dried skim milk in TBST (150 mM NaCl, 20 mM tris, 0.05 % Tween 20) for 2 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The specific antibodies were KPNA2 (mouse; 1:500; Santa Cruz Biotechnology, USA), MMP-1 (rabbit; 1:500; Santa Cruz Biotechnology, USA), MMP-13 (rabbit; 1:500; Santa Cruz Biotechnology, USA), P65 (rabbit; 1:500; Santa Cruz Biotechnology, USA), ACTIN (mouse; 1:1000; Santa Cruz Biotechnology, USA), and GAPDH (rabbit; 1:1000, Santa Cruz Biotechnology, USA). Thereafter, the membranes were washed with TBST three times and later incubated with the secondary antibody for 2 h at room temperature. The band density was measured with a computer-assisted image analysis system (Adobe Systems, San Jose, CA, USA) and normalized against ACTIN or GAPDH levels. Values are responsible for at least three independent reactions.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells Co-immunoprecipitation Assay SW982 cells were harvested and lysed in buffer (50 mmol/l Tris–HCl, pH 7.5, 150 mmol/l NaCl, 5 mmol/ l EDTA, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS). Thirty microliters of supernatants were collected as input. The remaining liquids were precleared with 30 μl protein A/G agarose (Santa Cruz) on a rocker at 4 °C for 2 h. After that, the precleared supernatants were separated into two and incubated with the 6 μl KPNA2 antibody (Santa Cruz) and 0.3 μl IgG, respectively, at 4 °C overnight with gentle agitation. Then, the samples were incubated with 30 μl protein A/G at 4 °C for 2 h with gentle agitation. At last, deposits were collected. The immune complexes were then analyzed by immunoblotting using specific antibodies against KPNA2 and P65. RT-PCR Total RNA was isolated from the SW982 cells using the TRIzol reagent (Invitrogen, Burlington, Ontario, Canada) according to the manufacturer’s protocol and then checked by agarose gel electrophoresis and ethidium bromide staining. PCR (30 cycles) was performed as follows: denaturation for 30 s annealing at 55 °C for 40 s and elongation at 72 °C for 40 s. Primers used for PCR were as follows: IL-6 forward 5′-GAGGAGACTTGCCTGG TGA-3′ and reverse 5′-TACATTTGCCGAAGAGCC-3′. PCR products were analyzed by agarose (2%) gel electrophoresis, and the bands were then visualized by ethidium bromide staining. The relative differences in expression between groups were expressed using optical density normalized with GAPDH. Analysis of results was based on five independent experiments. Sections and Immunohistochemistry Tissue sections from patient’s specimen were then cut at 5 um with a cryostat, and the sections were stored at −20 °C before use. Thereafter, all sections were processed in 10-mM citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 20 min to retrieve the antigen, and then, endogenous peroxidase activity was blocked by soaking in 3 % hydrogen peroxide. After rinsing in phosphatebuffered saline (PBS) (pH 7.2), the sections were incubated with the primary antibody against KPNA2 (mouse; 1:200; Santa Cruz Biotechnology, USA) for 2 h at room temperature. All slides were processed using the peroxidase–antiperoxidase method (DAKO, Hamburg, Germany). After rinsing in PBS, the peroxidase reaction was visualized by incubating the sections with the liquid mixture DAB
(0.02 % diaminobenzidine tetrahydrochloride, 0.1 % phosphate buffer solution, and 3 % H2O2). After rinsing in water, the sections were counterstained with hematoxylin, dehydrated, and cover slipped. Immunofluorescent Histochemical Staining Analysis Sections were blocked with 10 % normal serumblocking solution species, the same as the secondary antibody, containing 3 % (w/v) BSA and 0.1 % Triton X-100 and 0.05 % Tween 20 for 2 h at room temperature to avoid unspecific staining. Then, the sections were incubated with both an antibody specific for KPNA2 and an antibody for different markers: myeloperoxidase (MPO) (rabbit antiMPO, 1:100, Abcam), ED1 (rabbit anti-ED1, 1:100, Santa Cruz Biotechnology), CD3 (rabbit anti-CD3, 1:100, Abcam), thymocyte differentiation antigen 1 (THY1) (rabbit anti-THY1, 1:100, Santa Cruz Biotechnology), overnight at 4 °C. Briefly, sections were incubated with both primary antibodies overnight at 4 °C, followed by a mixture of fluorescein isothiocyanate (FITC)- and tetramethylrhodamine (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at 4 °C. The stained sections were examined under a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). Immunofluorescence Cell Staining Analysis SW982 cells were fixed with 4 % formaldehyde for 30 min, then treated with 0.1 % TritonX-100/phosphatebuffered saline (PBS) for 5 min, and incubated with PBS containing 3 % normal goat serum for 1 h. The cells were incubated overnight at 4 °C with an antibody specific for KPNA2 and anti-NF-κB P65 (Santa Cruz, USA). After washing the cells with PBS, they were incubated with FITC- and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch). To detect the nucleus,, the cells were counterstained with 4,6-diamidino-2phenylindole (DAPI; 0.1 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) for 40 min at 30 °C. The cells were rinsed and mounted onto slides, which were then analyzed and imaged by a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). siRNA and Transfection The target of KPNA2 siRNA was 5′-GCUGGU UUGAUUCCGAAAUTT-3′. SW982 cells were seeded the day before transfection using DMEM with 10 % FBS. Transient transfection was carried out
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang using Lipofectamine 2000 according to the manufacturer’s instructions [16], and cells were incubated 6 h at 37 °C in DMEM with no serum or antibiotics. The transfection mixtures were changed after 6 h to 10 % FBS-containing DMEM, and cells were cultured for further 48 h before use. The experiments were repeated at least three times.
Statistical Analysis All data were expressed as mean ± SEM. Data were compared using the Student’s t test. P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition.
RESULTS
Upregulation of KPNA2 Expression in Synovial Tissue of RA Patients Western blot was performed to investigate the expression of KPNA2 in synovial tissue of RA patients and healthy individuals. Statistical analysis revealed that protein expression of KPNA2 increased in RA patients compared with the healthy controls (P < 0.05) (Fig. 1a). Immunohistochemistry analysis was employed to identify the distribution of KPNA2 in synovial tissue of RA and healthy individuals. The synovial tissue sections of RA patients and healthy individuals were stained with antibodies against KPNA2. We found that the expression of
Fig. 1. Expression and distribution of KPNA2 protein in synovial tissue of RA and healthy individuals. a Expression of KPNA2 in synovial tissue of RA and healthy controls was determined by Western blot. Upper blot shows KPNA2 protein levels; lower blot demonstrates equal loading by detecting GAPDH. Bands were quantified by densitometer, and the amount of KPNA2 was normalized by referring to the amount of GAPDH. b Immunohistochemical detection of KPNA2. Health (left); RA (right). c Immunofluorescent detection of KPNA2. Health (left); RA (right). The Health group (n = 10) contained four males and six females; the mean age was 44.5 years. The RA group (n = 10) contained five males and five females; the mean age was 45.2 years. Compared with healthy groups (asterisk), P < 0.05, original magnification ×400 in b and c.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells KPNA2 was relatively overexpressed in synovial tissue obtained from RA patients compared with the healthy controls (Fig. 1b). By immunofluorescent staining of KPNA2, we observed that there were only a few KPNA2-positive cells in synovium tissue obtained from healthy controls, but many KPNA2-positive cells appeared in synovium of RA patients (Fig. 1c).
examined the co-localization of KPNA2 with CD3 (a marker for T cells), THY1 (a marker for fibroblast-like synoviocytes), MPO (a marker for neutrophils), and ED1 (a marker for macrophage-like synoviocytes). This examination revealed that KPNA2 co-localized with T cells (Fig. 2a), fibroblast-like synoviocytes (Fig. 2b), neutrophils (Fig. 2c), and macrophage-like synoviocytes (Fig. 2d) in synovial tissue of patients with RA.
Cellular Localization of KPNA2 in RA Synovium Increasing evidence suggests that the invasive monocytes/macrophages, lymphocytes, and resident fibroblastlike synoviocytes produce many cytokines and inflammatory mediators and play a role in the pathological progress in RA patients. To further determine the accurate cell types in which KPNA2 was expressed in the RA synovium, we performed the double immunofluorescent staining and
The Expression of KPNA2 was Increased in SW982 Cells and Primary FLS with TNFα Treatment Based on our studies in vivo, we speculated that KPNA2 might participate in the process of RA. To confirm this hypothesis, we performed in vitro experiments using the SW982 cells incubated with TNFα (10 ng/ml), a key proinflammatory cytokine that was involved in the
Fig. 2. Cellular localization of KPNA2 in the RA synovium. Immunofluorescent staining was performed on sections of synovial tissue of patients with RA. TRITC (red) signals indicate KPNA2. FITC (green) signals indicate CD3 (a), THY1 (b), MPO (c), and ED1 (d). Merge pictures (a–d) showed that KPNA2 co-localized in T cells, fibroblast-like synoviocytes, neutrophils, and macrophage-like synoviocytes in synovial tissue of RA patients (original magnification ×400).
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang
Fig. 3. KPNA2, MMP-1, MMP-13, and IL-6 expressions were remarkably upregulated in TNFα-induced SW982 cells. After incubating SW982 cells with serum-free DMEM for 24 h, TNFα was added into the media at a final concentration of 10 ng/ml. Cells were cultured for another 6, 12, 24, and 36 h. a KPNA2 and b MMP-1 and MMP-13 protein expressions were measured by Western blot assay. d IL-6 mRNA expressions were measured by RT-PCR. c KPNA2 and MMP-1 and MMP-13 protein expressions were measured by Western blot assay in primary cells induced by TNFα (10 ng/ml). Compared with untreated control cells (asterisk), P < 0.05.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells pathogenesis of RA. Western blot analysis presented that, following TNFα stimulation, the protein expression levels of KPNA2 were enhanced in a time-dependent manner and reached the peak at 24 h in SW982 cells (Fig. 3a). As predicted, TNFα significantly increased the expression of MMP-1 and MMP-13 proteins (Fig. 3b) and IL-6 mRNA (Fig. 3d) in a time-dependent manner in SW982 cells. In the meantime, we also confirmed our previous conclusion by Western blot in primary cells induced by TNFα (Fig. 3c). TNFα Induces Translocation of KPNA2 and P65 to the Nucleus in SW982 Cells NF-κB P65 subunit is a sign of NF-κB activation; so in our experiment, we take P65 as research objects [17]. Previous study reported that nuclear import of P65 is regulated by KPNA2 in a nuclear localization signal (NLS)dependent manner [18]. We then checked whether TNFα could affect the intracellular distribution of KPNA2 and P65 in synovial cells. Thereafter, SW982 cells were treated by TNFα for 0 to 48 h, separated into cytoplasmic and nuclear fractions, and analyzed by Western blot. As a result, the amount of NF-κB P65 and KPNA2 levels was in cycles of
up and down in the nucleus by treatment with TNFα at 30 min (Fig. 4a). To determine whether TNFα affects NFκB signaling pathway, we also examined the effect of TNFα treatment (0, 10, 30, 60 min) on the phosphorylation level of P65 by Western blotting. Within 10 min after addition of TNFα, the level of P-P65 in total cell lysate was greatly increased (Fig. 4b). These results suggest that TNFα induces translocation of KPNA2 and NF-κB to the nucleus. The Association of KPNA2 and P65 in SW982 Cells in RA To further assess the nuclear translocation and possible association of NF-κB P65 subunit and KPNA2 in the SW982 cells after TNFα treatment, double immunofluorescent staining was used. As shown in Fig. 5, KPNA2 and P65 immunoreactivity was evenly distributed throughout the cytoplasm but rarely detected in the nucleus in untreated SW982 cells (Fig. 5a, b). Incubation with 10 ng/ml TNFα caused a shift of NF-κB P65 and KPNA2 from cytoplasm towards the nucleus at 30 min (Fig. 5a, b). Double immunofluorescent staining revealed a clear colocalization of KPNA2 and P65 proteins both in the untreated cells and treated cells (Fig. 5c). In line with this, P65
Fig. 4. Time course of KPNA2 and P65 nuclear translocation in TNFα-treated SW982 cells. SW982 cells were stimulated with 10 ng/ml TNFα for the periods indicated. Whole cell or nuclear extracts were immunoblotted with anti-P-P65, anti-KPNA2, anti-P65, anti-Lamin B, and anti-Actin antibodies. Compared with untreated control cells (asterisk), P < 0.05).
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang
Fig. 5. Distribution and co-localization of P65 subunit and KPNA2 in TNFα-treated SW982 cells and RA tissues. The SW982 cells were treated with 10 ng/ ml TNFα for 30 min, and double immunofluorescent staining was performed with TRITC-labeled anti-KPNA2 antibody and FITC-labeled anti-P65 antibody and analyzed by microscopy. a and b presented the nuclear translocation of KPNA2 and P65 following TNFα administration. c and d indicated that KPNA2 co-localized with P65 in SW982 cells and in synovial tissues of RA. FITC (green) signals indicate P65; TRITC (red) signals indicate KPNA2 (original magnification ×400). KPNA2 and P65 interaction demonstrated by co-immunoprecipitation assay (e). SW982 cells were harvested for assay. The immune complexes were analyzed by Western blotting with the specific antibodies. All results are representatives of at least three repeated experiments.
and KPNA2 were co-localized with each other in the synovial tissues of RA patients (Fig. 5d). To further study the mechanism of KPNA2 in RA, co-immunoprecipitation was used to test the relationship of KPNA2 and P65. We found that KPNA2 interacted with P65 in SW982 cells (Fig. 5e). These results confirmed that KPNA2 binds to P65 and plays a major role in P65 import. Inhibition of KPNA2 Alleviated TNFα-Induced Inflammation in SW982 Cells To get a deeper understanding of the function of KPNA2 in RA, we knocked down the expression of endogenous KPNA2 by siRNA in TNFα-treated SW982 cells. Knockdown efficiency was confirmed by Western blot analysis (Fig. 6a). As shown in (Fig. 6b), inhibiting
KPNA2 expression significantly reduced TNFα-induced MMP-1 expression in SW982 cells compared with control siRNA. Furthermore, RT-PCR assay presented that knockdown of KPNA2 alleviated TNFα-stimulated IL-6 expression (Fig. 6c). More importantly, the KPNA2 knockdown greatly inhibited the phosphorylation of p65 following TNFα treatment (Fig. 6b). These observations implied that KPNA2 might promote the development of inflammation in synovial cells via a NF-κB p65-dependent pathway and thus participate the development of RA.
DISCUSSION The development of RA is characterized by an infiltration of immune cells into the synovium, leading
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells
Fig. 6. Knockdown of KPNA2 alleviated TNFα-induced inflammation in human SW982 cells. TNFα (10 ng/ml)-treated SW982 cells were transfected with KPNA2 siRNA, control siRNA, or vehicle for 48 h. Western blot was performed to confirm the efficiency of KPNA2 siRNA-mediated downregulation. The bar graph indicated the density of KPNA2 versus GAPDH. Western blot (b) and RT-PCR (c) showed that knocking KPNA2 down led to decreased levels of MMP-1 protein, P-P65 protein, and IL-6 mRNA in TNFα (10 ng/ml)-treated SW982 cells. Compared with the control group (asterisk), P < 0.05).
to chronic inflammation and pannus formation and eventually leading to cartilage and bone destruction [21]. The RA synovium consists of neutrophils, macrophages, T cells, synovial fibroblasts, and other synovial cells such as endothelium [19, 20]. IL-6, a B cell
regulatory factor, is likely the primary driver of the hepatic acute phase response in RA. IL-6 promotes synovitis by inducing neovascularisation via pannus proliferation, resulting in infiltration of inflammatory cells and synovial hyperplasia [21]. MMP-1 is one of
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang the critical neutral proteinases which degrade native fibrillar collagens in the extracellular matrix (ECM) [22]. Although the pathogenesis of RA is still not illustrated, it is well known that activation of NF-κB-dependent gene expression plays a key role in RA development. NFκB has long been appreciated as a central player in many of the processes underlying the progression of RA. NF-κB is comprised of a family of five DNA-binding proteins: relA (p65), relB, c-rel, p50, and p52. In the most canonical example, p50/relA dimers are held in the cytoplasm by IκBα. An inflammatory signal such as TNFα induces the activation of the IκB kinase (IKK) complex to phosphorylate members of the IκB family. Phosphorylated IκB becomes ubiquitinated and is then targeted for degradation by the proteasome. The NF-κB dimers can then translocate to the nucleus and activate the transcription and/or repression of genes. NF-κB has been implicated in cytokine release, activation of virtually all immune cells, osteoclast activation, autoantibody production, cellular proliferation, inhibition of apoptosis, and numerous other processes associated with RA [23, 24]. KPNA2, a member of the importin family, comprises an IBB domain, eight armadillo repeats that are involved in recognition and binding to nuclear localization signals, and a C-terminal acidic domain [25]. KPNA2 is a major component of nuclear pore complexes required for transporting various macromolecules larger than 50 KDa or other complex proteins [26, 27]. The nuclear transport signaling pathway was previously identified as important for tumorigenesis and tumor development [27]. However, there are no reports about the biological role for KPNA2 in RA. In the present study, we found that the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Then, our work showed that KPNA2 was co-localized with neutrophils, macrophagelike synoviocytes, fibroblast-like synoviocytes, and T cells. These cells play a key role in the development of the synovial inflammation seen in RA and result in destruction of cartilage and bone [28]. By making use of human synovial cell line SW982, we further confirmed that TNFα can significantly induce the synovial cell inflammatory reaction, which demonstrated by the production of MMP-1, MMP13, and IL-6. Consistent with the in vivo results, KPNA2 expression was significantly upregulated by TNFα in SW982 cells. The above results implied that KPNA2 might participate in the synovial inflammation of RA. NF-κB/p65 is retained in the cytoplasm until it is activated in response to stimulation. Nuclear import of p65 is regulated by importin α in a nuclear localization signal
(NLS)-dependent manner [20]. Prior research has shown that KPNA2 first binds to the NLS of the P65 protein and subsequently recruits importin β to its IBB domain at the N terminus [29]. The complexes are transported into the nucleus through nuclear pore complexes (NPCs) embedded on the nuclear membrane [29, 30]. Given the pivotal role of NF-κB signaling pathway in RA, we next focused on the possible association between KPNA2 and P65 subcellular translocation in SW982 cells and RA tissues. Following TNFα stimulation, both Western blot and immunofluorescent staining revealed the co-localization and translocation from cytoplasma to nucleus of KPNA2 and P65 in SW982 cells. Inhibiting KPNA2 expression with siRNA greatly alleviated TNFα-induced expression of MMP-1 and IL-6 and, more importantly, decreased the P65 phosphorylation. Thus, these results indicated that KPNA2 played an important role in the RA synovial inflammation processes via NFκB P65 pathway. Further study is needed to clarify the precise interaction between KPNA2 and P65, which would be a focus for a new treatment to regulate the inflammation process of RA.
ACKNOWLEDGMENTS This work was supported by Postgraduate Technology Innovation Project of Nantong University (YKS14004), Clinical Medicine Special Funds of Jiangsu Province (BL2014059), National Basic Research Program of China (973 Program, No. 2012CB822104), National Natural Science Foundation of China (31170766), National Natural Science Foundation of China (81171140), Key Project Natural Science Foundation of Jiangsu University and College (No. 11KJA310002), Nantong City Social Development Projects funds (HS2012032), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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KPNA2 Contributes to the Inflammatory Processes in Synovial Tissue of Patients with Rheumatoid Arthritis and SW982 Cells Zhongbing Liu,1 Dongmei Zhang,1,2 Chi Sun,1 Ran Tao,1 Xinbao Xu,1 Libin Xu,1 Hongbing Cheng,3 Min Xiao,4 and Youhua Wang1,5
Abstract—Karyopherin-α2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. We investigated the function and possible mechanisms of KPNA2 involved in rheumatoid arthritis (RA). Western blotting and immunohistochemistry showed the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Double immunofluorescent staining indicated that KPNA2 co-localized with T cells, macrophage-like synoviocytes, fibroblast-like synoviocytes, and neutrophils in synovial tissue of RA patients. Moreover, the expression of KPNA2 in SW982 cells was increased in a time-dependent manner in response to TNFα stimulation. Both Western blotting and immunofluorescent staining assay revealed the co-localization of KPNA2 and P65 and their translocation from cytoplasma in TNFα-treated SW982 cells. Furthermore, knocking down the expression of KPNA2 by siRNA inhibited TNFα-induced expression of IL-6, MMP-1, and MMP-13 and, more importantly, decreased the P65 phosphorylation in SW982 cells. We therefore suggested that KPNA2 may play a key role in the inflammation process of RA via NF-κB P65 signal transduction pathway. KEY WORDS: karyopherin-α2 (KPNA2); rheumatoid arthritis (RA); SW982; tumor necrosis factor-α (TNFα); P65.
INTRODUCTION Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease which is characterized by infiltration of inflammatory cell and resulting in synovial hyperplasia and joint destruction [1, 2]. The pathogenesis of proliferation has still not been clarified, but the characteristics of Zhongbing Liu and Dongmei Zhang contributed equally to this work. 1
Department of Orthopaedics, Affiliated Hospital of Nantong University, and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001Jiangsu Province, China 2 Department of Pathogen Biology, Medical College, Nantong University, Nantong, 226001, China 3 Department of Orthopaedics, Traditional Chinese Medical Hospital of Nantong City, Nantong, 226001, China 4 National Glycoengineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China 5 To whom correspondence should be addressed at Department of Orthopaedics, Affiliated Hospital of Nantong University, and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001Jiangsu Province, China. E-mail: [email protected]
RA synoviocytes are similar to those of transformed cells [3]. NF-κB, one of the major transcription factor implicated in joint inflammation, is essential for the production of cytokines and proteases by RA fibroblast-like synoviocytes (FLSs), and it mediates the resistance of RA FLS to apoptosis [4–6]. In humans, the synovial cell is a major source of inflammatory cytokines such as IL-1, IL-6, and TNFα, all of which play a chief role during the development of RA [7]. Because of these cytokines, NFκB can be activated and further cause the expression of various inflammatory mediators. Karyopherin-α2 (KPNA2), a member of the importin family, is a kind of nuclear transport proteins [8]. One of the last major steps of the most intracellular signaling pathways is nucleocytoplasmic transport [9]. Some proteins such as transcription factors in the cytoplasm enter the nucleus and modulate the expression of the effect genes or other signaling molecules. Nucleocytoplasmic transport of the molecules within the cytoplasm occurs through large nuclear pore complexes in the nuclear membrane [10]. Preliminary studies showed that KPNA2 was associated
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang with cell proliferation and tumor formation, and people paid more and more attention to its importance [11]. However, the expression of KPNA2 and its biological function in RA remains to be elucidated. The human synovial cell line, SW982, is a useful tool to investigate the expression of inflammatory cytokines or MMPs in response to TNFα [12, 13]. In this study, we first investigated the expression of KPNA2 in synovial tissue of patients with RA and SW982 cells and explored the mechanism involved in the RA pathogenesis.
MATERIALS AND METHODS Patients and RASFs There were five females and five males in our cohort of RA patients. Samples were obtained from patients at the time of total knee replacement surgery. All patients were diagnosed according to the standards defined by the American College of Rheumatology in 1987. The disease activity was calculated by using the disease activity score (DAS), and, for the current study only, patients with a DAS higher than 3.2 were included [14, 15]. The synovial tissue from ten healthy individuals was isolated during arthroscopic procedures performed by orthopedic surgeons as control. Age in the control group was 45±5 years and for the RA patients was 46±6 years. The study was approved by the institutional medical ethics committee of the Affiliated Hospital of Nantong University and informed consent was obtained from all patients prior to surgery. Cell Culture and Stimulation Synovial tissue was digested for 2 h with 0.25 % (weight/volume) collagenase and then suspended in RPMI 1640 medium with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in 5 % CO2 for several days, after which nonadherent cells were removed. RASFs from passages 3 to 7 were used for our experiment. The human synovial cell line SW982 was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in humidified air with 5 % CO2. The medium was changed every 2–3 days, and cell passage was carried out every 5–6 days. For in vitro experiments in SW982 cells, cells were incubated with serum-free DMEM for 24 h and then stimulated with
TNFα (Human, Sigma Chemical Co., St. Louis, MO, USA) at the indicated concentration and time. The cells were harvested after treatment and used for further analysis. Western Blot Analysis The synovial tissue samples of RA patients and healthy individuals were homogenized in lysis buffer (1 % NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l ethylene diamine tetraacetic acid (EDTA), 1 % sodium dodecyl sulfate (SDS), 1 % sodium deoxycholate, 1 % Triton X-100, 1 mmol/l phenylmethyl sulfonyl fluoride (PMSF), 10 lg/ml aprotinin, and 1 lg/ml leupeptin) and then centrifuged at 12,000 and 4 rpm for 20 min to collect the supernatant. Cell cultures for immunoblot were lysed in buffer (10 mM tris, pH 8.0,150 mM NaCl, 1 % nonidet P-40, 0.5 % deoxycholate, 0.1 % SDS and protease inhibitor mixture) followed by centrifugation to discard cell debris. To prepare cytoplasmic and nuclear extracts of cells, we homogenized frozen samples in the above buffer, then centrifuged them at 14,000 rpm for 1 min at 4 °C, and finally stored cytoplasmic extracts at −80 °C at once. We resuspended nuclei in a low-salt buffer and agitated. Samples were centrifuged at 14,000 rpm for 5 min at 4 °C, and nuclear extracts were stored at −80 °C immediately. After determining the protein concentration with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene diflouride filter (PVDF) membranes (Millipore, Bed ford, MA, USA). The membranes were blocked with 5 % dried skim milk in TBST (150 mM NaCl, 20 mM tris, 0.05 % Tween 20) for 2 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The specific antibodies were KPNA2 (mouse; 1:500; Santa Cruz Biotechnology, USA), MMP-1 (rabbit; 1:500; Santa Cruz Biotechnology, USA), MMP-13 (rabbit; 1:500; Santa Cruz Biotechnology, USA), P65 (rabbit; 1:500; Santa Cruz Biotechnology, USA), ACTIN (mouse; 1:1000; Santa Cruz Biotechnology, USA), and GAPDH (rabbit; 1:1000, Santa Cruz Biotechnology, USA). Thereafter, the membranes were washed with TBST three times and later incubated with the secondary antibody for 2 h at room temperature. The band density was measured with a computer-assisted image analysis system (Adobe Systems, San Jose, CA, USA) and normalized against ACTIN or GAPDH levels. Values are responsible for at least three independent reactions.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells Co-immunoprecipitation Assay SW982 cells were harvested and lysed in buffer (50 mmol/l Tris–HCl, pH 7.5, 150 mmol/l NaCl, 5 mmol/ l EDTA, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS). Thirty microliters of supernatants were collected as input. The remaining liquids were precleared with 30 μl protein A/G agarose (Santa Cruz) on a rocker at 4 °C for 2 h. After that, the precleared supernatants were separated into two and incubated with the 6 μl KPNA2 antibody (Santa Cruz) and 0.3 μl IgG, respectively, at 4 °C overnight with gentle agitation. Then, the samples were incubated with 30 μl protein A/G at 4 °C for 2 h with gentle agitation. At last, deposits were collected. The immune complexes were then analyzed by immunoblotting using specific antibodies against KPNA2 and P65. RT-PCR Total RNA was isolated from the SW982 cells using the TRIzol reagent (Invitrogen, Burlington, Ontario, Canada) according to the manufacturer’s protocol and then checked by agarose gel electrophoresis and ethidium bromide staining. PCR (30 cycles) was performed as follows: denaturation for 30 s annealing at 55 °C for 40 s and elongation at 72 °C for 40 s. Primers used for PCR were as follows: IL-6 forward 5′-GAGGAGACTTGCCTGG TGA-3′ and reverse 5′-TACATTTGCCGAAGAGCC-3′. PCR products were analyzed by agarose (2%) gel electrophoresis, and the bands were then visualized by ethidium bromide staining. The relative differences in expression between groups were expressed using optical density normalized with GAPDH. Analysis of results was based on five independent experiments. Sections and Immunohistochemistry Tissue sections from patient’s specimen were then cut at 5 um with a cryostat, and the sections were stored at −20 °C before use. Thereafter, all sections were processed in 10-mM citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 20 min to retrieve the antigen, and then, endogenous peroxidase activity was blocked by soaking in 3 % hydrogen peroxide. After rinsing in phosphatebuffered saline (PBS) (pH 7.2), the sections were incubated with the primary antibody against KPNA2 (mouse; 1:200; Santa Cruz Biotechnology, USA) for 2 h at room temperature. All slides were processed using the peroxidase–antiperoxidase method (DAKO, Hamburg, Germany). After rinsing in PBS, the peroxidase reaction was visualized by incubating the sections with the liquid mixture DAB
(0.02 % diaminobenzidine tetrahydrochloride, 0.1 % phosphate buffer solution, and 3 % H2O2). After rinsing in water, the sections were counterstained with hematoxylin, dehydrated, and cover slipped. Immunofluorescent Histochemical Staining Analysis Sections were blocked with 10 % normal serumblocking solution species, the same as the secondary antibody, containing 3 % (w/v) BSA and 0.1 % Triton X-100 and 0.05 % Tween 20 for 2 h at room temperature to avoid unspecific staining. Then, the sections were incubated with both an antibody specific for KPNA2 and an antibody for different markers: myeloperoxidase (MPO) (rabbit antiMPO, 1:100, Abcam), ED1 (rabbit anti-ED1, 1:100, Santa Cruz Biotechnology), CD3 (rabbit anti-CD3, 1:100, Abcam), thymocyte differentiation antigen 1 (THY1) (rabbit anti-THY1, 1:100, Santa Cruz Biotechnology), overnight at 4 °C. Briefly, sections were incubated with both primary antibodies overnight at 4 °C, followed by a mixture of fluorescein isothiocyanate (FITC)- and tetramethylrhodamine (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at 4 °C. The stained sections were examined under a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). Immunofluorescence Cell Staining Analysis SW982 cells were fixed with 4 % formaldehyde for 30 min, then treated with 0.1 % TritonX-100/phosphatebuffered saline (PBS) for 5 min, and incubated with PBS containing 3 % normal goat serum for 1 h. The cells were incubated overnight at 4 °C with an antibody specific for KPNA2 and anti-NF-κB P65 (Santa Cruz, USA). After washing the cells with PBS, they were incubated with FITC- and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch). To detect the nucleus,, the cells were counterstained with 4,6-diamidino-2phenylindole (DAPI; 0.1 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) for 40 min at 30 °C. The cells were rinsed and mounted onto slides, which were then analyzed and imaged by a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). siRNA and Transfection The target of KPNA2 siRNA was 5′-GCUGGU UUGAUUCCGAAAUTT-3′. SW982 cells were seeded the day before transfection using DMEM with 10 % FBS. Transient transfection was carried out
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang using Lipofectamine 2000 according to the manufacturer’s instructions [16], and cells were incubated 6 h at 37 °C in DMEM with no serum or antibiotics. The transfection mixtures were changed after 6 h to 10 % FBS-containing DMEM, and cells were cultured for further 48 h before use. The experiments were repeated at least three times.
Statistical Analysis All data were expressed as mean ± SEM. Data were compared using the Student’s t test. P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition.
RESULTS
Upregulation of KPNA2 Expression in Synovial Tissue of RA Patients Western blot was performed to investigate the expression of KPNA2 in synovial tissue of RA patients and healthy individuals. Statistical analysis revealed that protein expression of KPNA2 increased in RA patients compared with the healthy controls (P < 0.05) (Fig. 1a). Immunohistochemistry analysis was employed to identify the distribution of KPNA2 in synovial tissue of RA and healthy individuals. The synovial tissue sections of RA patients and healthy individuals were stained with antibodies against KPNA2. We found that the expression of
Fig. 1. Expression and distribution of KPNA2 protein in synovial tissue of RA and healthy individuals. a Expression of KPNA2 in synovial tissue of RA and healthy controls was determined by Western blot. Upper blot shows KPNA2 protein levels; lower blot demonstrates equal loading by detecting GAPDH. Bands were quantified by densitometer, and the amount of KPNA2 was normalized by referring to the amount of GAPDH. b Immunohistochemical detection of KPNA2. Health (left); RA (right). c Immunofluorescent detection of KPNA2. Health (left); RA (right). The Health group (n = 10) contained four males and six females; the mean age was 44.5 years. The RA group (n = 10) contained five males and five females; the mean age was 45.2 years. Compared with healthy groups (asterisk), P < 0.05, original magnification ×400 in b and c.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells KPNA2 was relatively overexpressed in synovial tissue obtained from RA patients compared with the healthy controls (Fig. 1b). By immunofluorescent staining of KPNA2, we observed that there were only a few KPNA2-positive cells in synovium tissue obtained from healthy controls, but many KPNA2-positive cells appeared in synovium of RA patients (Fig. 1c).
examined the co-localization of KPNA2 with CD3 (a marker for T cells), THY1 (a marker for fibroblast-like synoviocytes), MPO (a marker for neutrophils), and ED1 (a marker for macrophage-like synoviocytes). This examination revealed that KPNA2 co-localized with T cells (Fig. 2a), fibroblast-like synoviocytes (Fig. 2b), neutrophils (Fig. 2c), and macrophage-like synoviocytes (Fig. 2d) in synovial tissue of patients with RA.
Cellular Localization of KPNA2 in RA Synovium Increasing evidence suggests that the invasive monocytes/macrophages, lymphocytes, and resident fibroblastlike synoviocytes produce many cytokines and inflammatory mediators and play a role in the pathological progress in RA patients. To further determine the accurate cell types in which KPNA2 was expressed in the RA synovium, we performed the double immunofluorescent staining and
The Expression of KPNA2 was Increased in SW982 Cells and Primary FLS with TNFα Treatment Based on our studies in vivo, we speculated that KPNA2 might participate in the process of RA. To confirm this hypothesis, we performed in vitro experiments using the SW982 cells incubated with TNFα (10 ng/ml), a key proinflammatory cytokine that was involved in the
Fig. 2. Cellular localization of KPNA2 in the RA synovium. Immunofluorescent staining was performed on sections of synovial tissue of patients with RA. TRITC (red) signals indicate KPNA2. FITC (green) signals indicate CD3 (a), THY1 (b), MPO (c), and ED1 (d). Merge pictures (a–d) showed that KPNA2 co-localized in T cells, fibroblast-like synoviocytes, neutrophils, and macrophage-like synoviocytes in synovial tissue of RA patients (original magnification ×400).
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang
Fig. 3. KPNA2, MMP-1, MMP-13, and IL-6 expressions were remarkably upregulated in TNFα-induced SW982 cells. After incubating SW982 cells with serum-free DMEM for 24 h, TNFα was added into the media at a final concentration of 10 ng/ml. Cells were cultured for another 6, 12, 24, and 36 h. a KPNA2 and b MMP-1 and MMP-13 protein expressions were measured by Western blot assay. d IL-6 mRNA expressions were measured by RT-PCR. c KPNA2 and MMP-1 and MMP-13 protein expressions were measured by Western blot assay in primary cells induced by TNFα (10 ng/ml). Compared with untreated control cells (asterisk), P < 0.05.
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells pathogenesis of RA. Western blot analysis presented that, following TNFα stimulation, the protein expression levels of KPNA2 were enhanced in a time-dependent manner and reached the peak at 24 h in SW982 cells (Fig. 3a). As predicted, TNFα significantly increased the expression of MMP-1 and MMP-13 proteins (Fig. 3b) and IL-6 mRNA (Fig. 3d) in a time-dependent manner in SW982 cells. In the meantime, we also confirmed our previous conclusion by Western blot in primary cells induced by TNFα (Fig. 3c). TNFα Induces Translocation of KPNA2 and P65 to the Nucleus in SW982 Cells NF-κB P65 subunit is a sign of NF-κB activation; so in our experiment, we take P65 as research objects [17]. Previous study reported that nuclear import of P65 is regulated by KPNA2 in a nuclear localization signal (NLS)dependent manner [18]. We then checked whether TNFα could affect the intracellular distribution of KPNA2 and P65 in synovial cells. Thereafter, SW982 cells were treated by TNFα for 0 to 48 h, separated into cytoplasmic and nuclear fractions, and analyzed by Western blot. As a result, the amount of NF-κB P65 and KPNA2 levels was in cycles of
up and down in the nucleus by treatment with TNFα at 30 min (Fig. 4a). To determine whether TNFα affects NFκB signaling pathway, we also examined the effect of TNFα treatment (0, 10, 30, 60 min) on the phosphorylation level of P65 by Western blotting. Within 10 min after addition of TNFα, the level of P-P65 in total cell lysate was greatly increased (Fig. 4b). These results suggest that TNFα induces translocation of KPNA2 and NF-κB to the nucleus. The Association of KPNA2 and P65 in SW982 Cells in RA To further assess the nuclear translocation and possible association of NF-κB P65 subunit and KPNA2 in the SW982 cells after TNFα treatment, double immunofluorescent staining was used. As shown in Fig. 5, KPNA2 and P65 immunoreactivity was evenly distributed throughout the cytoplasm but rarely detected in the nucleus in untreated SW982 cells (Fig. 5a, b). Incubation with 10 ng/ml TNFα caused a shift of NF-κB P65 and KPNA2 from cytoplasm towards the nucleus at 30 min (Fig. 5a, b). Double immunofluorescent staining revealed a clear colocalization of KPNA2 and P65 proteins both in the untreated cells and treated cells (Fig. 5c). In line with this, P65
Fig. 4. Time course of KPNA2 and P65 nuclear translocation in TNFα-treated SW982 cells. SW982 cells were stimulated with 10 ng/ml TNFα for the periods indicated. Whole cell or nuclear extracts were immunoblotted with anti-P-P65, anti-KPNA2, anti-P65, anti-Lamin B, and anti-Actin antibodies. Compared with untreated control cells (asterisk), P < 0.05).
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang
Fig. 5. Distribution and co-localization of P65 subunit and KPNA2 in TNFα-treated SW982 cells and RA tissues. The SW982 cells were treated with 10 ng/ ml TNFα for 30 min, and double immunofluorescent staining was performed with TRITC-labeled anti-KPNA2 antibody and FITC-labeled anti-P65 antibody and analyzed by microscopy. a and b presented the nuclear translocation of KPNA2 and P65 following TNFα administration. c and d indicated that KPNA2 co-localized with P65 in SW982 cells and in synovial tissues of RA. FITC (green) signals indicate P65; TRITC (red) signals indicate KPNA2 (original magnification ×400). KPNA2 and P65 interaction demonstrated by co-immunoprecipitation assay (e). SW982 cells were harvested for assay. The immune complexes were analyzed by Western blotting with the specific antibodies. All results are representatives of at least three repeated experiments.
and KPNA2 were co-localized with each other in the synovial tissues of RA patients (Fig. 5d). To further study the mechanism of KPNA2 in RA, co-immunoprecipitation was used to test the relationship of KPNA2 and P65. We found that KPNA2 interacted with P65 in SW982 cells (Fig. 5e). These results confirmed that KPNA2 binds to P65 and plays a major role in P65 import. Inhibition of KPNA2 Alleviated TNFα-Induced Inflammation in SW982 Cells To get a deeper understanding of the function of KPNA2 in RA, we knocked down the expression of endogenous KPNA2 by siRNA in TNFα-treated SW982 cells. Knockdown efficiency was confirmed by Western blot analysis (Fig. 6a). As shown in (Fig. 6b), inhibiting
KPNA2 expression significantly reduced TNFα-induced MMP-1 expression in SW982 cells compared with control siRNA. Furthermore, RT-PCR assay presented that knockdown of KPNA2 alleviated TNFα-stimulated IL-6 expression (Fig. 6c). More importantly, the KPNA2 knockdown greatly inhibited the phosphorylation of p65 following TNFα treatment (Fig. 6b). These observations implied that KPNA2 might promote the development of inflammation in synovial cells via a NF-κB p65-dependent pathway and thus participate the development of RA.
DISCUSSION The development of RA is characterized by an infiltration of immune cells into the synovium, leading
KPNA2 Mediate Inflammation from Synovial Tissue and SW982 Cells
Fig. 6. Knockdown of KPNA2 alleviated TNFα-induced inflammation in human SW982 cells. TNFα (10 ng/ml)-treated SW982 cells were transfected with KPNA2 siRNA, control siRNA, or vehicle for 48 h. Western blot was performed to confirm the efficiency of KPNA2 siRNA-mediated downregulation. The bar graph indicated the density of KPNA2 versus GAPDH. Western blot (b) and RT-PCR (c) showed that knocking KPNA2 down led to decreased levels of MMP-1 protein, P-P65 protein, and IL-6 mRNA in TNFα (10 ng/ml)-treated SW982 cells. Compared with the control group (asterisk), P < 0.05).
to chronic inflammation and pannus formation and eventually leading to cartilage and bone destruction [21]. The RA synovium consists of neutrophils, macrophages, T cells, synovial fibroblasts, and other synovial cells such as endothelium [19, 20]. IL-6, a B cell
regulatory factor, is likely the primary driver of the hepatic acute phase response in RA. IL-6 promotes synovitis by inducing neovascularisation via pannus proliferation, resulting in infiltration of inflammatory cells and synovial hyperplasia [21]. MMP-1 is one of
Liu, Zhang, Sun, Tao, Xu, Xu, Cheng, Xiao, and Wang the critical neutral proteinases which degrade native fibrillar collagens in the extracellular matrix (ECM) [22]. Although the pathogenesis of RA is still not illustrated, it is well known that activation of NF-κB-dependent gene expression plays a key role in RA development. NFκB has long been appreciated as a central player in many of the processes underlying the progression of RA. NF-κB is comprised of a family of five DNA-binding proteins: relA (p65), relB, c-rel, p50, and p52. In the most canonical example, p50/relA dimers are held in the cytoplasm by IκBα. An inflammatory signal such as TNFα induces the activation of the IκB kinase (IKK) complex to phosphorylate members of the IκB family. Phosphorylated IκB becomes ubiquitinated and is then targeted for degradation by the proteasome. The NF-κB dimers can then translocate to the nucleus and activate the transcription and/or repression of genes. NF-κB has been implicated in cytokine release, activation of virtually all immune cells, osteoclast activation, autoantibody production, cellular proliferation, inhibition of apoptosis, and numerous other processes associated with RA [23, 24]. KPNA2, a member of the importin family, comprises an IBB domain, eight armadillo repeats that are involved in recognition and binding to nuclear localization signals, and a C-terminal acidic domain [25]. KPNA2 is a major component of nuclear pore complexes required for transporting various macromolecules larger than 50 KDa or other complex proteins [26, 27]. The nuclear transport signaling pathway was previously identified as important for tumorigenesis and tumor development [27]. However, there are no reports about the biological role for KPNA2 in RA. In the present study, we found that the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Then, our work showed that KPNA2 was co-localized with neutrophils, macrophagelike synoviocytes, fibroblast-like synoviocytes, and T cells. These cells play a key role in the development of the synovial inflammation seen in RA and result in destruction of cartilage and bone [28]. By making use of human synovial cell line SW982, we further confirmed that TNFα can significantly induce the synovial cell inflammatory reaction, which demonstrated by the production of MMP-1, MMP13, and IL-6. Consistent with the in vivo results, KPNA2 expression was significantly upregulated by TNFα in SW982 cells. The above results implied that KPNA2 might participate in the synovial inflammation of RA. NF-κB/p65 is retained in the cytoplasm until it is activated in response to stimulation. Nuclear import of p65 is regulated by importin α in a nuclear localization signal
(NLS)-dependent manner [20]. Prior research has shown that KPNA2 first binds to the NLS of the P65 protein and subsequently recruits importin β to its IBB domain at the N terminus [29]. The complexes are transported into the nucleus through nuclear pore complexes (NPCs) embedded on the nuclear membrane [29, 30]. Given the pivotal role of NF-κB signaling pathway in RA, we next focused on the possible association between KPNA2 and P65 subcellular translocation in SW982 cells and RA tissues. Following TNFα stimulation, both Western blot and immunofluorescent staining revealed the co-localization and translocation from cytoplasma to nucleus of KPNA2 and P65 in SW982 cells. Inhibiting KPNA2 expression with siRNA greatly alleviated TNFα-induced expression of MMP-1 and IL-6 and, more importantly, decreased the P65 phosphorylation. Thus, these results indicated that KPNA2 played an important role in the RA synovial inflammation processes via NFκB P65 pathway. Further study is needed to clarify the precise interaction between KPNA2 and P65, which would be a focus for a new treatment to regulate the inflammation process of RA.
ACKNOWLEDGMENTS This work was supported by Postgraduate Technology Innovation Project of Nantong University (YKS14004), Clinical Medicine Special Funds of Jiangsu Province (BL2014059), National Basic Research Program of China (973 Program, No. 2012CB822104), National Natural Science Foundation of China (31170766), National Natural Science Foundation of China (81171140), Key Project Natural Science Foundation of Jiangsu University and College (No. 11KJA310002), Nantong City Social Development Projects funds (HS2012032), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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