Free Radical Biology and Medicine 76 (2014) 251–260

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Original Contribution

Knockdown of peroxiredoxin 5 inhibits the growth of osteoarthritic chondrocytes via upregulating Wnt/β-catenin signaling Yini Ma, Rongheng Li n, Yudi Zhang, Lingyun Zhou, Yehong Dai Department of Combination of Chinese and Western Medicine, The First Affiliated Hospital of Chongqing Medical University, You Yi Road 1#, Chongqing 400016, People’s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 1 June 2014 Received in revised form 12 August 2014 Accepted 13 August 2014 Available online 16 September 2014

Peroxiredoxin 5 is a member of the peroxiredoxin family, which has been shown to act as an antioxidant whose main function is to reduce reactive oxygen species in cells. Peroxiredoxin 5 has been found to be abnormally elevated in human osteoarthritic chondrocytes. However, the detailed mechanism by which peroxiredoxin 5 modulates human osteoarthritic chondrocytes’ survival has not been elucidated. In the current study, we demonstrated that peroxiredoxin 5 knockdown activated osteoarthritic chondrocytes apoptosis, and decreased scavenging of endogenous reactive oxygen species. Furthermore, silencing of peroxiredoxin 5 resulted in an altered expression of proteins associated with Wnt signaling. Collectively, these results demonstrated that the regulatory effects of peroxiredoxin 5 can be partially attributed to Wnt/β-catenin signaling. & 2014 Elsevier Inc. All rights reserved.

Keywords: Peroxiredoxin 5 Osteoarthritis Apoptosis Reactive oxygen species Cartilage Chondrocytes

Introduction Osteoarthritis (OA) is the most prevalent articular disorder and it is increasingly becoming a major cause of disability in aged people. Multiple factors are believed to cause OA, such as trauma, abnormal mechanical loading, failure of nutrient supply, and genetic predisposition [1]. Current available drugs to treat OA are predominantly directed toward the symptomatic relief of pain and inflammation but they do little to reduce joint destruction [2]. Therefore, the discovery of molecules essential to the initiation and progression of OA as well as new therapeutic strategies to treat OA are important for improving the prognosis and therapy of OA patients. The pathological processes responsible for OA initiation and progression are very complex and poorly understood, despite the extensive research efforts into this complex disease. Several studies have revealed that reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2), hydroxyl radicals, and nitric oxide can contribute to the onset and progression of OA by inducing chondrocyte death and matrix degradation [3–5]. To prevent

Abbreviations: Prdx5, peroxiredoxin 5; ROS, reactive oxygen species; OA, osteoarthritis; H2O2, hydrogen peroxide; SOD, superoxide dismutase; Prdxs, peroxiredoxins; DMEM, Dulbecco’s modified Eagle’s medium; NAC, N-acetyl-L-cysteine; 7-AAD, 7-amino-actinomycin D; MTT, 3-(4, 5-dimethylthiozol-2yl)-2, 5-diphenyltrazolium bromide n Corresponding author. E-mail address: [email protected] (R. Li). http://dx.doi.org/10.1016/j.freeradbiomed.2014.08.015 0891-5849/& 2014 Elsevier Inc. All rights reserved.

toxicity by ROS, chondrocytes possess a well coordinated antioxidant enzyme system formed by superoxide dismutase (SOD), catalase, and glutathione peroxidase. Recently, a novel family of peroxidases, the peroxiredoxins (Prdxs), was identified in many living organisms. Six isoforms of Prdxs have been identified in mammals, all of which participate directly in eliminating H2O2 and neutralizing other oxidizing chemicals. Peroxiredoxin 5 (Prdx5) is a thioredoxin peroxidase which is highly expressed in many tissues [6]. Like other 2-Cys peroxiredoxins, Prdx5 requires a thioredoxin [7] as a reducing partner. Prdx5 is upregulated in degenerative human tendon and its overexpression protects human tendon cells against apoptosis [8,9]. Normal human cartilage also constitutively expresses Prdx5 [10,11]. Moreover, Prdx5 is found to be elevated in OA cartilage and its expression is upregulated by IL-1 and TNF-α. This IL-1/TNF-α stimulating effect is fully inhibited by catalase, indicating that H2O2 might be an important mediator for the cytokine-induced Prdx5 upregulation. It has been suggested that Prdx5 may play a protective role against oxidative stress involved in the pathogenesis of OA, and may have therapeutic value in the prevention and treatment of OA [12]. Despite these insights, the biological functions of Prdx5 in osteoarthritic chondrocytes have not been fully characterized, and the underlying mechanisms remain poorly understood. Wnt signaling constitutes one of the most critical biological processes during cell fate assignment and homeostasis [13]. It is essential for bone homeostasis and its effects in chondrogenic differentiation and cartilage formation are complex [14]. Recent studies further suggest the relevance of Wnt signaling in human

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OA [15–17]. Because Wnt pathways are involved in both cartilage and bone formation that include a role in regulation of chondrocyte hypertrophy in the growth plate, dysregulation of Wnt pathways in adult tissues could contribute to the chondrocyte hypertrophy seen in OA and pathologic changes in cartilage and bone [18]. Wnt/β-catenin signaling is described as part of the canonical Wnt pathway [19]. β-Catenin is a key element in driving Wnt/β-catenin signaling [20]. In humans, 10 frizzled receptors and at least 19 Wnt proteins are known. For instance, Frizzled-2 is expressed in synovial tissue of arthritic cartilage [21]; it also influences cell proliferation and the response to activating stimuli. Elevated expression of Frizzled-2 has been linked to tissue regeneration and hyperplasia in an animal model of atherosclerosis [22]. Wnt-4 is expressed within developing joints [23,24] and it also has been found to be differentially expressed during specific stages of mesenchymal condensation and/or chondrocyte differentiation [24–27]. Furthermore, hyperexpression of Wnt-4 blocks mesenchymal condensation [28]. The Wnt protein binds to Frizzled family receptors and low density lipoprotein receptor-related protein (LRP) 5/6 can activate the Wnt signaling pathway, which results in the activation of dishevelled (Dvl) family proteins. The activation of Dvl leads to the inhibition of glycogen synthase kinase (GSK)-3β (the enzyme responsible for proteosomal β-catenin degradation). Without Wnt signaling, GSK-3β is thought to phosphorylate and consequently induce the degradation of β-catenin, thus keeping intracellular β-catenin levels low [29]. When the kinase activity of GSK-3β is suppressed, nonphosphorylated β-catenin can accumulate in the cytoplasm, since it is no longer continuously phosphorylated and degraded by the proteosome. The accumulating β-catenin migrates to the nucleus, where it binds to transcription factors, such as lymphoid enhancing factor/ T-cell factor, to generate a transcriptionally active complex that targets genes such as Myc, cyclin D1, matrix metalloproteinase MMP-3, and CD44 [30]. Increased levels of β-catenin have been observed in degenerative cartilage, suggesting that a diminished capacity to limit Wnt signaling might contribute to cartilage loss [31–33]. In recent studies, ROS have been implicated in the regulation of Wnt signaling [34,35]. Several research works have shown that treatment of cells with H2O2 to induce ROS-dependent signaling inhibits β-catenin/TCF transcriptional activity [36,37]. In addition, a recent study indicated that Wnt/β-catenin signaling is involved in increased apoptosis and decreased proliferation in colorectal cancer cells by Prdx2 knockdown [38]. Based on the above research results, we speculated that the regulatory role of Prdx5, in the survival of osteoarthritic chondrocytes, may be associated with the Wnt/β-catenin signaling. In this study, we investigated the expression of Prdx5 in human normal and osteoarthritic chondrocytes, and examined if Prdx5 plays a role in the survival of osteoarthritic chondrocytes and whether these effects are mediated by the Wnt/β-catenin signaling.

Materials and methods Reagents and antibodies Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand Island, NY). Collagenase, dimethyl sulfoxide (DMSO), 3-(4, 5-dimethylthiozol-2yl)-2, 5-diphenyltrazolium bromide (MTT), and XAV-939 were from Sigma–Aldrich (St. Louis, MO, USA). pTOPflash reporter plasmid was obtained from Upstate Biotechnology (NY, USA). Fetal bovine serum (FBS) was obtained from HyClone (Shanghai, China). Annexin V-PE and a 7-AAD (7-amino-actinomycin D) double-staining apoptosis detection kit and Total protein extraction kit were purchased from KeyGEN

(Nanjing, China). Reactive oxygen species assay kit and N-acetyl-Lcysteine (NAC) were purchased from Beyotime (Jiangsu, China). PrimeScript RT Reagent kit, Trizol, and SYBR Premix Ex Taq II were purchased from TaKaRa (Dalian, China). Pierce BCA protein assay kit was from Pierce Biotechnology (USA). Lentiviral constructs expressing Prdx5 shRNA (Prdx5-shRNA-LV) were purchased from Shanghai Genechem Co., Ltd., China. The Prdx5 and Frizzled-2 polyclonal rabbit antibody were purchased from Abcam (UK). The Wnt-4, β-catenin, GSK-3β, p-GSK-3βser9, MMP-13, cyclin D1, Lamin B1, and β-actin antibodies were purchased from Epitomics (CA, USA). Alexa Fluor 488-conjugated goat anti-rabbit IgG was from ZSGB-BIO (Beijing, China). Tissue specimens and histologic evaluation OA cartilage samples (n ¼8) were obtained from joints of patients with OA undergoing knee replacement surgery at the Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University. Human normal articular cartilage samples were obtained from 8 patients with knee joint fractures undergoing orthopedic operations at the Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, and excluded patients with a history of joint disease. The study was approved by the Medical Ethics Review Committee of The First Affiliated Hospital of Chongqing Medical University. Informed consent was given by all patients involved. Research has been performed in accordance with the Declaration of Helsinki involving human material. For histological analysis, samples were fixed in 4% paraformaldehyde, decalcified, and then processed through a series of increasing ethanol concentrations for dehydration. These samples were subsequently paraffin-embedded, and 4 μm sections were stained with hemotoxylin and eosin (HE). Histological scoring of the cartilage was performed by two blinded investigators and determined according to the Mankin scoring system [39]. Cell culture For explant cultures and chondrocytes, the articular cartilage was minced and digested in 0.15% (w/v) collagenase in DMEM supplemented with 10% FBS, 2% penicillin/streptomycin (Beyotime, Jiangsu, China) for 16 h at 37 1C as described previously [40]. The digest was centrifuged and the cells were resuspended in FBS-enriched DMEM and cultured in flasks at 37 1C in 5% CO2. Subcultures were performed with 0.25% trypsin ethylenediaminetetraacetic acid (Gibco) and first passage cells were used. Construction of Prdx5-shRNA lentiviral vector and transduction of chondrocytes with shRNA Three pairs of specific shRNA sequences which target the three sites of Prdx5 coding sequences and one pair of negative control oligonucleotide sequence were cloned into the lentiviral expression vector pMU6-MCS-Ubi-EGFP plasmid (restricted by HpaI and XhoI). The sequence of three siRNAs candidates were as follows: si-001, sequence GGA ATC GAC GTC TCA AGA GGT; si-002, sequence CCA CTC TTG AGA CGT CGA CAA; si-003, sequence GCC TTG AGA CGT CAT CGA TGA. Bacterial clones with insertions of siRNA oligonucleotides were identified using PCR and sequenced with vector primers GCC CCG GTT AAT TTG CAT AT (forward) and CAC CCA AGA TCT GGC CTC (reverse). After identification by restriction and sequencing, the pMU6-MCS-Ubi-EGFP vector plasmid and two helper plasmids pHelper1.0 and pHelper2.0 were cotransfected into the 293T cells, and the lentiviral supernatant particles were packaged. Osteoarthritic chondrocytes and normal chondrocytes were seeded in 6-well plates at a concentration of

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0.5  105 cells per well (20–30% confluence) on the day before shRNA transduction. Prdx5-shRNA-lentivirus was transduced into chondrocytes at a multiplicity of infection (MOI) of 40 using polybrene (10 mg/ml) and Enhanced Infection Solution (Genechem, China). At the same time, a negative control virus GFP-lentivirus (Genechem, China) was transduced into chondrocytes using the same methods to control for the impact of the viral vector. After incubation for 12 h, the medium was replaced with fresh DMEM. Transduction effects were observed with a fluorescence microscopy camera 72 h after transduction. After the indicated time points, the cells were harvested for subsequent studies.

Chondrocytes viability assay Cell viability was evaluated using the MTT assay. For this assay, 20 ml of MTT [5 mg/ml in phosphate buffered saline (PBS)] was added to each well and the plate was incubated for four additional hours at 37 1C. Thereafter, 150 ml DMSO was added to each well to dissolve the water-insoluble formazan salt with shaking for 10 min. For NAC-response experiments, cells were pretreated with different concentrations of NAC (1.0–5 mM) for 24 h. The optical density values were measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Flow cytometry analysis An Annexin V-PE and 7-AAD (7-amino-actinomycin D) doublestaining apoptosis detection kit was used to detect apoptotic activity according to the manufacturer’s instructions. All experiments were performed three times. After the indicated treatments, the cells were subjected to fluorescence-activated cell sorting (FACS) analysis within an hour using CellQuest software version 3.3.

Detection of intracellular ROS A ROS assay kit was used to measure intracellular ROS according to the manufacturer’s instructions. The transduced osteoarthritic chondrocytes were incubated in 10 mM DCFH-DA containing medium at 37 1C for 25 min. For XAV-939-response experiments, cells were pretreated with XAV-939 (5 mM) for 24 h. Cells were then washed with PBS three times to remove DCFH-DA that had not entered the cells and resuspended in DMEM medium, and the fluorescence intensity of the cell suspension was immediately measured by FACS analysis.

Immunofluorescence assay Immunofluorescence was used to detect expression of β-catenin in transduced cells. Cells were seeded and cultured on glass coverslips the day prior to analysis. After incubation for 24 h, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After fixation, the cells were permeabilized with 0.2% Triton X-100 (Beyotime, Jiangsu, China) and then blocked with 10% normal goat serum for 1 h at room temperature. The cells were then incubated with rabbit polyclonal antibodies specific for β-catenin (1:200) simultaneously overnight at 4 1C. After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 488conjugated goat anti-rabbit IgG) for 1 h at 37 1C. Nuclei were counterstained with DAPI (KeyGEN Biotech, Nanjing, China) for 10 min, and images were obtained using an Olympus microscope (Olympus Corporation, Tokyo, Japan).

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Quantitative real-time PCR Total RNA was isolated from the osteoarthritic chondrocytes immersed in Trizol as described by the manufacturer. Total RNA was reverse-transcribed using the PrimeScript RT Reagent kit. Real-time PCRs were performed using SYBR Premix Ex Taq II according to the manufacturer’s instructions in a CFX96 RealTime System (Bio-Rad, USA). The primers for Prdx5, MMP-13, and cyclin D1 were as follows: Prdx5 (forward 50 -ATC AGC CAG GAG CCG AAC C-30 and reverse 50 -GTC CGC AGT TTC AGC AGA GC-30 ); MMP-13 (forward 50 -GGA GCA TGG CGA CTT CTA C-30 and reverse 50 - GAG TGC TCC AGG GTC CTT-30 ); and cyclin D1 (forward 50 - AAT GCC AGA GGC GGA TGA GA -30 and reverse 50 - GCT TGT GCG GTA GCA GGA GA -30 ). β-Actin was used as an internal control. The relative expression levels of mRNAs were calculated using the Δ Δ 2  ( Ct sample– Ct control) method. Western blotting Proteins in the OA cartilage tissue homogenates or osteoarthritic chondrocytes and normal chondrocytes lysate were isolated using a Total protein extraction kit according to the manufacturer’s instructions. Moreover, to detect the cellular localization of β-catenin, nuclear and cytoplasmic fractions were isolated using a nuclear and cytoplasmic protein extraction kit (KeyGEN, Nanjing, China) according to the manufacturer’s instructions. The protein extracts (50 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were then incubated with rabbit polyclonal antibodies specific for Prdx5 (1:2000), Wnt-4 (1:1000), GSK-3β (1:1000), β-catenin (1:1000), p-β-cateninser33/37 (1:2000), p-GSK-3βser9 (1:1000), Frizzled-2 (1:1000), MMP-13(1:1000), and cyclin D1(1:3000) overnight at 4 1C. After washing with PBS containing 0.05% Tween 20, the membranes were incubated with secondary antibody (1:3000) for 2 h. The membranes were then visualized using chemiluminescence detection reagents according to the manufacturer’s instructions and an enhanced chemiluminescence detection system (ChemiDoc XRS imager, Bio-Rad, USA) and β-actin and Lamin B1 were used as the internal control. Reporter assays To determine the transcriptional activity of β-catenin, transient transfections were performed with Lipofectamine 2000 transfection reagent for the TOPflash plasmid containing three copies of the β-catenin/T-cell factor (TCF)-binding sites upstream of a minimal herpesvirus thymidine kinase promoter driving the firefly luciferase expression. Thirty thousand cells were plated in 24-well plates 30 min before the addition of a mixture containing 20 μl of serumfree DMEM, 0.6 μl of Lipofectamine 2000, 0.4 μg of the TOPflash reporter construct, 0.8 μg of Prdx5-shRNA-lentivirus, and 0.8 μg of the Renilla luciferase vector phRG-TK (Upstate Biotechnology). Cells were subjected to culture for 24 h and lysed in 50 μl of lysis buffer, and the luciferase activity was determined with a luminometer using the Dual Luciferase Assay system kit (Promega) using 20 μl of lysates. The firefly luciferase activity was normalized to the activity of the renilla luciferase. The activity of the TOPflash reporter construct was expressed as normalized relative luminescence units. Statistical analysis Statistical analysis software [Statistical Package for the Social Sciences (SPSS) version 19.0, IBM, NY, USA] was used for all statistical tests. All data, displayed as the mean7standard deviation (SD), were analyzed by analysis of variance (ANOVA) and Student’s t tests.

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Differences were considered significant at P value o0.05. All experiments were repeated in triplicate.

Results Prdx5 and MMP-13 protein are upregulated in osteoarthritic cartilage compared with normal articular cartilage We evaluated 8 tissue specimens from OA patients by quantitative real-time PCR (qRT-PCR) and Western blotting assays for Prdx5 expression. The results showed that Prdx5 mRNA and protein were highly expressed in cartilage, and steady-state levels of Prdx5 protein were 2.3-fold higher in OA cartilage than in normal articular cartilage

samples (Po0.05, Fig. 1A), whereas mRNA levels of Prdx5 were 2.5-fold higher in OA cartilage than in normal articular cartilage samples (Po0.05, Fig. 1B). The results were similar to the results from the previous research studied by Min-Xia Wang and his colleagues [12], suggesting an upregulation of Prdx5 in OA. Because MMP-13 plays an important role in the pathological process of osteoarthritis, we then evaluated the expression of MMP-13 protein in tissue specimens from OA patients by Western blotting assays, and the results indicated significantly elevated expression levels for MMP13 in OA cartilage compared to the normal articular cartilage samples (Po0.05, Fig. 1C). Furthermore, we analyzed the histological scoring of articular cartilage. It was seen that the total Mankin score for OA cartilage was significantly increased compared to the normal articular cartilage samples (Po0.05, Table 1).

Fig. 1. Prdx5 is highly expressed in human OA articular cartilage. (A) The expression of Prdx5 in human OA articular cartilage and human normal articular cartilage was assayed by Western blotting. The expression of β-actin was used as a loading control. Prdx5 protein expression was significantly increased in OA articular cartilage compared to normal articular cartilage (n¼ 8, three replicates, *P o0.05 vs normal articular cartilage). (B) The expression of Prdx5mRNA in human OA articular cartilage and human normal articular cartilage was assayed by qRT-PCR analysis. Prdx5 expression was significantly increased in the OA articular cartilage compared with the normal articular cartilage (n ¼8, three replicates, *P o 0.05, **Po 0.01 vs normal articular cartilage). (C) The expression of MMP-13 in human OA articular cartilage and human normal articular cartilage was assayed by Western blotting. The expression of β-actin was used as a loading control. The results indicated significantly elevated expression levels for MMP-13 in OA cartilage compared to the normal articular cartilage (n¼ 8, three replicates, *P o 0.05, **Po 0.01 vs normal articular cartilage).

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Prdx5 expression is suppressed by Prdx5 siRNA in osteoarthritic chondrocytes As the Prdx5 protein is extremely abundant in osteoarthritic chondrocytes, we propose that it may play a crucial role in OA development and apoptosis. The siRNAs targeting the Prdx5 gene were synthesized to silence its expression. The effects of Prdx5 siRNA were studied using qRT-PCR and Western blot analysis. The results showed that the expression of Prdx5 at both the mRNA and the protein levels in osteoarthritic chondrocytes transfected with the Prdx5 shRNA was significantly lower than in osteoarthritic chondrocytes transfected with the negative control shRNA as determined by qRT-PCR and Western blotting analysis, respectively. The most effective siRNA sequence, si-001, was used for further investigation (P o0.05, Fig. 2A and B).

Table 1 Histological Mankin scoring of articular cartilage samples.

Structure Cells Toluidine staining Tidemark integrity Total score

Normal (n ¼8)

OA (n¼ 8)

0.28 7 0.02 0.53 7 0.05 0.38 7 0.04 0.197 0.01 1.38 7 0.13

5.197 0.87* 2.077 0.35* 3.417 0.49* 0.727 0.11* 11.39 7 2.06*

Data are expressed as mean 7 SD. n

Po 0.05 vs the normal articular cartilage.

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Prdx5 knockdown induces osteoarthritic chondrocyte apoptosis To determine the effects of Prdx5 shRNA on cell viability, the number of surviving chondrocytes following treatment was measured using the MTT assay. As shown in Fig. 3A, the number of surviving chondrocytes was decreased following transfection with Prdx5 shRNA compared to that of control transfected chondrocytes. Moreover, to establish if Prdx5 knockdown led to enhanced apoptosis, the apoptosis ratios in chondrocytes treated with Prdx5 shRNA were analyzed by flow cytometry. The results showed that the rate of apoptosis in chondrocytes treated with Prdx5 shRNA was increased more than 4-fold when compared to that of the control (P o0.05, Fig. 3B). Suppression of Prdx5 leads to decreased scavenging of ROS in osteoarthritic chondrocytes Because Prdx5 is known to function as an antioxidant enzyme, we investigated if suppression of Prdx5 increased intracellular levels of ROS. As shown in Fig. 3C, the DCF fluorescence intensity in osteoarthritic chondrocytes transduced with Prdx5 shRNA was more than twice as high as that in the negative control-transfected osteoarthritic chondrocytes, as measured by FACS analysis. To identify whether the increased level of apoptosis observed in silenced osteoarthritic chondrocytes was due to an increase in ROS, we further evaluated the viability and apoptosis in silenced osteoarthritic chondrocytes following the addition of the antioxidant NAC. After treatment of silenced osteoarthritic chondrocytes with different concentrations of NAC (1.5–5 mM) for 24 h, the results indicated

Fig. 2. Prdx5 expression is suppressed by Prdx5 shRNA in osteoarthritic chondrocytes. (A) Prdx5 protein expression in osteoarthritic chondrocytes transfected with Prdx5 shRNA (Si-Prdx5) was significantly lower than in osteoarthritic chondrocytes transfected with the negative control shRNA (Mock), or with the polybrene and Enhanced Infection Solution only (Control), as determined by Western blotting analysis. (Data are expressed as means 7 SD of three experiments. *P o 0.05 vs controls.) (B) Prdx5mRNA expression was markedly suppressed in osteoarthritic chondrocytes after transfection with Prdx5 shRNA (Si-Prdx5) compared with the expression in osteoarthritic chondrocytes transfected with the negative control shRNA (Mock), or with the polybrene and Enhanced Infection Solution only (Control), as shown by qRT-PCR analysis. (Data are expressed as means 7 SD of three experiments. **P o0.01 vs controls).

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Fig. 3. Effects of Prdx5 knockdown on osteoarthritic chondrocytes viability, apoptosis, and endogenous production of ROS. (A) Osteoarthritic chondrocytes viability was markedly inhibited after transfection with Prdx5 shRNA in comparison to osteoarthritic chondrocytes transfected with the negative control shRNA (Mock), or with the polybrene and Enhanced Infection Solution only (Control), as determined by the MTT assay. (Data are expressed as means 7 SD of three experiments. *Po 0.05 vs controls.) (B) The apoptosis ratio was significantly increased after a 72-h transfection with Prdx5 shRNA in comparison to osteoarthritic chondrocytes transfected with the negative control shRNA (Mock), or with the polybrene and Enhanced Infection Solution only (Control), as determined by FACS analysis. (Data are expressed as means 7 SD of three experiments. **P o 0.01 vs controls.) (C) DCFH-DA probe staining showed that transfection with Prdx5 shRNA osteoarthritic chondrocytes resulted in significant ROS accumulation in osteoarthritic chondrocytes. DCF fluorescence was increased more than 2-fold in osteoarthritic chondrocytes transfected with Prdx5 shRNA compared to the Mock group and Control group chondrocytes. Data are expressed as means 7 SD of three experiments. **P o 0.01 vs controls.) (D and E) Viability and apoptosis were evaluated in silenced chondrocytes treated with the antioxidant NAC by MTT assay and FACS analysis, respectively. The results showed significant enhancement of viability and decreased apoptosis in silenced chondrocytes treated with NAC compared to silenced chondrocytes treated without NAC. (Data are expressed as means 7 SD of three experiments. *P o 0.05, **Po 0.01 vs controls).

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significantly increased viability and decreased apoptosis in NACtreated silenced osteoarthritic chondrocytes compared to untreated silenced chondrocytes (Fig. 3D and E). This suggested that increased intracellular ROS, which is induced by suppression of Prdx5, induces apoptosis in osteoarthritic chondrocytes. Suppression of Prdx5 promotes the nuclear translocation of β-catenin, decreases GSK-3β activity, and enhances β-catenin/Tcf-dependent transcription in osteoarthritic chondrocytes To further determine the effects of suppression of Prdx5 on the Wnt/β-catenin signaling pathway in osteoarthritic chondrocytes, we focused on the effect of Prdx5 shRNA treatment on the intracellular localization of β-catenin, the activity of GSK-3β, and the transcriptional activity of target genes. Following Prdx5 silencing, the levels of intranuclear β-catenin were significantly increased in osteoarthritic chondrocytes. In addition, the levels of phosphorylated β-catenin (Ser33/37), which generates the degradation signal (without Wnt stimulation, β-catenin is constantly degraded by the proteosome, and this degradation strictly depends on β-catenin phosphorylation [41]), were significantly decreased following Prdx5 silencing (Po 0.05, Fig. 4A). However, the total expression levels of β-catenin were largely unchanged (Fig. 4A). We further evaluated the activity of GSK-3β, which is known to play a critical role in Wnt/β-catenin signaling. The levels of GSK-3β were significantly decreased in Prdx5-silenced osteoarthritic chondrocytes, whereas the levels of phosphorylated GSK-3β (Ser9) were significantly increased in Prdx5-silenced osteoarthritic chondrocytes (Po0.05, Fig. 4A). Moreover, we also examined the protein expression of Wnt-4 and Frizzled-2. Following Prdx5 silencing, the levels of Wnt-4 and Frizzled-2 were significantly increased in osteoarthritic chondrocytes treated with Prdx5 shRNA compared to the negative control-transfected osteoarthritic chondrocytes (P o0.05, Fig. 4A). In addition, the promoting effects of Prdx5 silencing on the expression of cyclin D1 and MMP-13 were evidenced by the upregulation of cyclin D1 and MMP-13 mRNA and protein expression levels in osteoarthritic chondrocytes following transduction with Prdx5 shRNA (P o0.05, Fig. 4A and B). Furthermore, the β-catenin/Tcf-dependent transcriptional activity was significantly enhanced in osteoarthritic chondrocytes following Prdx5 silencing (P o0.05, Fig. 4C). However, the Wnt/β-catenin signaling was not significantly affected in normal chondrocytes after transfection with Prdx5 shRNA. It is indicated that the effect of Prdx5 on Wnt/β-catenin signaling is a consequence of OA inflammation (Fig. 4D). We next evaluated changes in the intranuclear location of β-catenin in osteoarthritic chondrocytes using immunofluorescence. As shown in Fig. 5, β-catenin was observed to translocate from its location in the cytoplasm in negative control-transduced osteoarthritic chondrocytes to the nucleus in silenced osteoarthritic chondrocytes. Taken together, our results indicated that Prdx5 may be involved in the regulation of Wnt/β-catenin signaling in osteoarthritic chondrocytes. XAV-939 reduces endogenous production of ROS in osteoarthritic chondrocytes To further study whether Prdx5 participates in the growth of osteoarthritic chondrocytes via the Wnt/β-catenin pathway, osteoarthritic chondrocytes were treated with the highly specific Wnt/β-catenin pathway inhibitor XAV-939. XAV-939 (5 mM) alone reduced endogenous production of ROS in osteoarthritic chondrocytes (Po0.05, Fig. 6). However, treatment with XAV-939 in osteoarthritic chondrocytes with Prdx5 knockdown did not further reduce the endogenous production of ROS compared to osteoarthritic chondrocytes treated with XAV-939 alone (Fig. 6).

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These results suggested that Prdx5 may play a protective role against oxidative stress involved in the pathogenesis of OA through the regulation of the Wnt/β-catenin pathway.

Discussion The previous study has reported that human chondrocytes express Prdx5 and the expression is significantly higher in osteoarthritic cartilage compared with normal cartilage [12]. However, the biological functions of Prdx5 in osteoarthritic chondrocytes have not been fully characterized, and the precise molecular mechanisms have not yet been elucidated. In view of this, our purposes were to evaluate the effects of Prdx5 gene silencing on osteoarthritic chondrocyte growth and establish the associated signaling mechanisms. In this study, we investigated the Prdx5 expression in osteoarthritic cartilage tissues by qRT-PCR and Western blotting assays. The results indicated that osteoarthritic cartilage tissues consistently expressed significantly higher Prdx5 levels than normal. Our current results further confirmed the results of the previous study [12]. Several previous studies have found that Prdxs are involved not only in oxidative stress protection mechanisms but also in cell differentiation [42], proliferation [43], apoptosis [44,45], and signal transduction [46]. Furthermore, to determine whether Prdx5 contributed to maintaining the survival of osteoarthritic chondrocytes, we silenced the Prdx5 gene in osteoarthritic chondrocytes by Prdx5-shRNA-LV transduction. Suppression of the Prdx5 gene using shRNA resulted in a decrease in Prdx5 expression in osteoarthritic chondrocytes. All Prdxs participate directly in eliminating H2O2 and neutralizing other oxidizing molecules [47]. But Prdx5 may have a broader activity against ROS compared with other isoforms of Prdx and other antioxidant enzymes [48,49]. We observed that the level of ROS in Prdx5 silenced osteoarthritic chondrocytes was greater than that in control osteoarthritic chondrocytes. Therefore, we inferred that inhibition of osteoarthritic chondrocytes growth following Prdx5 silenced was associated with an increase in cell death via apoptosis induced by decreased scavenging of ROS. The NAC-response experiments further confirmed the above assumption. The Wnt/β-catenin signaling pathway is required for normal development and it plays an important role in a number of cellular events, such as cell proliferation, migration, and differentiation. Many studies have demonstrated that the Wnt/β-catenin signaling pathway is closely related to cartilage function, including cartilage development and chondrocyte differentiation, and plays an important role in the progression of OA [50,51]. Inhibition of the Wnt signaling pathway has a protective effect on joint destruction in OA [52]. The pivotal event of Wnt/β-catenin signaling is the nuclear translocation of β-catenin to form a complex with intranuclear LEF/ TCF, which activates transcription of target genes [53]. In the present study, our results revealed that Prdx5 knockdown upregulated expression of Wnt-4 and Frizzled-2, decreased GSK-3β activity, promoted β-catenin translocation into the nucleus, and decreased the levels of β-catenin phosphorylation, thus resulting in significant upregulation of transcription of the LEF/TCF target genes cyclin D1. It was demonstrated that suppression of Prdx5 in osteoarthritic chondrocytes activated the Wnt/β-catenin signaling pathway by reducing the activity of GSK-3β, and promoting β-catenin translocation into the nucleus, thus inhibiting chondrocyte proliferation and inducing chondrocyte apoptosis. However, the Wnt/β-catenin signaling was not significantly affected in normal chondrocytes after transfection with Prdx5 shRNA. It is indicated that the effect of Prdx5 on Wnt/β-catenin signaling is a consequence of OA inflammation. MMP-13 has been shown to be critical for OA progression, and is upregulated or inappropriately activated during OA by stress-,

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Fig. 4. Suppression of Prdx5 promotes the nuclear translocation of β-catenin, decreases GSK-3β activity, and enhances β-catenin/Tcf-dependent transcription in osteoarthritic chondrocytes. (A) Osteoarthritic chondrocytes were transfected with the negative control shRNA (Mock), Prdx5 shRNA (Si-Prdx5), or with the polybrene and Enhanced Infection Solution only (Control) for 72 h, the subcellular localization of β-catenin protein and activity of GSK-3β were assessed by Western blotting assays. Representative immunoblot showing the upregulated expression of proteins associated with the Wnt signaling, including Wnt-4, nuclear β-catenin, Frizzled-2, p-GSK-3βser9, and cyclin D1 in the Prdx5 shRNA-transfected osteoarthritic chondrocytes. Lamin B1 (nuclear expression) and β-actin (cytoplasmic expression) were used as the loading controls. (Data are expressed as means 7SD of three experiments. *Po 0.05, **Po 0.01 vs controls.) (B) The expression levels of mRNA for cyclin D1 and MMP-13 were assessed at 72 h after transduction with Prdx5 shRNA by qRT-PCR analysis. (Data are expressed as means 7 SD of three experiments. *Po 0.05, **Po 0.01 vs controls.) (C) To determine the effect of Prdx5 shRNA on the transcriptional activity of β-catenin, cells were transfected with Prdx5 shRNA or negative control shRNA (Mock) and TOP-FLASH plasmids. After 24 h, the cells were lysed and used for the luciferase activity. The firefly luciferase activity was normalized to the activity of the renilla luciferase. The activity of the TOPFLASH reporter construct was expressed as normalized relative light units. (Data are expressed as means 7 SD of three experiments. *P o 0.05 vs controls.) (D) Normal chondrocytes were transfected with the negative control shRNA (Mock), Prdx5 shRNA (Si-Prdx5), or with the polybrene and Enhanced Infection Solution only (Control) for 72 h. The expression of proteins associated with the Wnt/β-catenin signaling pathway was assessed by Western blotting assays. The results showed that the Wnt/β-catenin signaling was not significantly affected in normal chondrocytes after transfection with Prdx5 shRNA. (Data are expressed as means 7 SD of three experiments. *P o0.05 vs controls).

inflammation-, and differentiation-induced signals [54]. Wnt/β-catenin signaling pathways have also been shown to mediate the expression of MMP-13 during OA progression [55,56]. In Prdx5

silenced osteoarthritic chondrocytes, the upregulation of the Wnt/ β-catenin signaling pathways led to upregulation of MMP-13. Upregulated MMP-13 cleaved components of the extracellular matrix,

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Fig. 5. Intracellular distribution of β-catenin in osteoarthritic chondrocytes after Prdx5 silencing. Immunofluorescence staining was performed at 72 h after transduction with Prdx5 shRNA or the negative control (200  ). The analysis showed that β-catenin translocated from the cytoplasm in the osteoarthritic chondrocytes transfected with the negative control to the nucleus in osteoarthritic chondrocytes transfected with Prdx5 shRNA.

compared with human normal articular cartilage. Prdx5 knockdown by RNA interference activated osteoarthritic chondrocytes apoptosis, which was mediated through decreased scavenging of endogenous ROS as well as positive regulation of Wnt/β-catenin signaling. Our findings suggest that Prdx5 might play a protective role in human osteoarthritic cartilage degeneration. However, further studies are needed to confirm and extend these preliminary findings.

Conflict of interest Fig. 6. XAV-939 reduces endogenous production of ROS in osteoarthritic chondrocytes. Osteoarthritic chondrocytes were treated with Prdx5 shRNA vectors and/or 5 mM XAV-939 to evaluate endogenous production of ROS in osteoarthritic chondrocytes. (Data are expressed as means 7 SD of three experiments. *P o 0.05 vs nontreated osteoarthritic chondrocytes).

accelerating the destruction of articular cartilage, and thus inducing apoptosis of osteoarthritic chondrocytes. We further found that inhibition of the Wnt/β-catenin pathway with XAV-939 in osteoarthritic chondrocytes with Prdx5 shRNA did not reduce the endogenous production of ROS of these cells to a greater extent. Thus, downregulation of Prdx5 leads to upregulation of the Wnt/β-catenin pathway and its downstream targets (cyclin D1 and MMP-13), decreased ROS scavenging, and ultimately inhibits the growth of osteoarthritic chondrocytes. In conclusion, our study has demonstrated that the antioxidant enzyme Prdx5 is upregulated in human osteoarthritic cartilage

The authors declare that they have no conflict of interests.

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