Peroxiredoxin 4 inhibits IL-1b-induced chondrocyte apoptosis via PI3K/AKT signaling


Background: Chondrocytes apoptosis induced by reactive oxygen species (ROS) plays a critical role in the pathogenesis of osteoarthritis (OA). Peroxiredoxin 4 (PRDX4), a member of the PRDX family, is essential for removing metabolic free radicals and reducing intracellular ROS. In this study, we sought to investigate the roles of PRDX4 on interleukin 1b (IL-1b)-induced chondrocyte apoptosis.

Methods: Primary chondrocytes were isolated from the articular cartilage of Sprague-Dawley rats, infected with PRDX4 overexpressing lentivirus and treated with IL-1b (10 ng/mL). Cell apoptosis and ROS production identified by flow cytometry. Protein expression levels was evaluated by Western blotting analysis. Nitric oxide (NO) production and Caspase-3/9 activation were assessed by the Griess reaction
method and colorimetric assay kit, respectively.

Results: PRDX4 overexpression in chondrocytes significantly decreased IL-1b-induced apoptosis. It also reversed the activity of IL-1b that increased ROS and NO production. PRDX4 overexpression reversed the activity of IL-1b that reduced the levels of Bcl-2, p-AKT and p-PRAS40, as well as increased Bax levels and Caspase-3/9 activation. More importantly, pre-treated with AKT inhibitor (AZD5363) significantly reduced the protective effects of PRDX4.

Conclusions: Our data demonstrated that the regulatory effects of PRDX4 on IL-1b-induced chondrocyte apoptosis can be partially attributed to phosphatidylinositol 3-kinase/AKT signaling. These results indicate that PRDX4 might play a protective role in OA cartilage degeneration.

1. Introduction

Osteoarthritis (OA) is a common degenerative joint disorder especially in elderly individuals. It is characterized by damaged articular cartilage, subchondral bone changes and synovial inflam- mation [1]. The pathogenesis of OA involves reactive oxygen species (ROS) overproduction and the altered redox signaling pathways [2]. Chondrocytes are the sole cell type in articular cartilage [3]. Studies have demonstrated that chondrocytes plays an important role in the maintenance of the articular cartilage matrix [4]. Blood vessels are absent in cartilage. As a result, chondrocytes are living underhypoxic conditions. In pathological conditions, excessive nitric oxide (NO) and other ROS are produced, which causes oxidative damage,induces chondrocyte apoptosis and cartilage degradation, and contributes to the onset and progression of OA [5–8].

Previous studies have indicated that pro-inflammatory cyto- kines secreted by chondrocytes, such as interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a), are implicated in OA pathophys- iology [9,10]. For instance, IL-1b levels are significantly higher in the synovial fluid and cartilage of OA patients than in that of controls [11]. Type I IL-1 receptor (IL-1R) is significantly increased in OA chondrocytes and synovial cells, making these cells more sensitivity to the stimulation of IL-1b [9,10]. Exposure of chondrocytes to IL-1b increases NO release [12], ROS production, mitochondrial damage, and ultimate cell apoptosis [13]. Numerous studies have reported that phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) is inactivated in IL-1b-mediated chondrocyte apoptosis [14–16]. Activated PI3K/AKT pathway was involved in the regulation of chondrocyte survival and proteoglycan synthesis [17,18]. Therefore, the PI3K/Akt signaling pathway is thought to participate in OA progression.

Peroxiredoxin 4 (PRDX4) belongs to a recently discovered antioxidant family. To date, 6 PRDX family members have been identified and characterized in mammals, all of which eliminate hydrogen peroxide and neutralize other oxidizing chemicals, thus protecting cells against oxidative stress[19]. PRDX4 can metabolize hydrogen peroxide produced by endoplasmic oxidoreductin (ERO1) in HT1080 human fibrosarcoma cells [20]. Overexpression of PRDX4 protects against apoptosis by ameliorating oxidative stress-induced injury in a model of type 1 diabetes mellitus [21], and a model of nonalcoholic steatohepatitis and type 2 diabetes [22]. In glioblasto- ma cells, suppression of PRDX4 can increase ROS and cell apoptosis [23]. A recentstudyshowed that PRDX5 knockdown in osteoarthritic chondrocytes resulted in an alteredexpression of proteinsassociated with Wnt signaling, induced cell apoptosis and reduced scavenging of endogenous ROS [24]. PRDX6 could protect retinal pigment epithelium cells against oxidative damage via PI3K/AKT signaling [25]. However, it remains unclear whether PRDX4 plays a role in ROS production and apoptosis of chondrocytes and whether PI3K/AKT signaling is involved in such process.

In this study, we investigated the effects of ectopic expression of PRDX4 in IL-1b-induced chondrocyte apoptosis. We also explored the mechanism how PRDX4 exerted a protective role by experi- ments with an AKT specific inhibitor.

2. Materials and methods

2.1. Isolation, primary culture and identification of chondrocytes

Cartilage tissues were collected from knee joints of 6-week-old Sprague-Dawley rats (Shanghai Experimental Animal Center, Shanghai, China), minced into less than 1 mm small pieces, and digested with 0.4% Type II collagenase solution (Sigma, St. Louis, MO, USA) at 37 ◦C for 5 h. The undigested tissues were filtered out with a 70-mm cell strainer. After centrifugation at 1000 rpm for 5 min, rat chondrocytes were isolated and stained with trypan blue to evaluate cell viability. The chondrocytes with a viability greater than 85% were maintained in Dulbecco’s Modified Eagles Medium (DMEM) medium containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and appropriate antibiotics at 37 ◦C in 5% CO2- containing humidified air.

The chondrocytes were cultured in coverslip for 24 h and fixed with 4% paraformaldehyde under room temperature for 30 min. After washing with PBS, cells were incubated with 3% H2O2 and then blocked with normal goat serum. Collagen II (Abcam, Cambridge, UK) or SOX9 (Abcam) antibody was applied accordingly at 4 ◦C for overnight and then incubated with secondary antibody for 20 min. The signals were developed with DAB agents and then stained with hematoxylin. Pictures were taken under the microscope at ×200 magnification.

2.2. Lentivirus production

To create pLVX-puro/PRDX4 expression construct, rat PDRX4 gene was synthesized by Genewiz (Beijing, China), ligated into the lentiviral vector pLVX-puro (Clontech, Palo Alto, CA, USA) and then verified by sequencing. pLVX-puro/PRDX4 or pLVX-puro together with viral packaging plasmids was transfected into HEK293T cells using lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA) per the manufacture’s protocol. PRDX4 expressing or Vector viral supernatant was harvested at 48 h after transfection, filtered through 0.45-mm filters and stored in aliquots at —80 ◦C.

2.3. Experimental grouping

The chondrocytes were divided into four groups: Group 1 (Control), cells were cultured without any treatment; Group 2, cells were infected with Vector virus for 24 h and then cultured for 24 h with 10 ng/mL IL-1b (Peprotech; Rocky Hill, NJ, USA); Group 3, cells were infected with PRDX4 expressing virus for 24 h and then cultured for 24 h with 10 ng/mL IL-1b; Group 4, cells were infected with PRDX4 expressing virus, pre-treated with 3 mM AZD5363 (Selleck; Houston, TX, USA) for 2 h and then cultured for 24 h with 10 ng/mL IL-1b.

2.4. Flow cytometric analysis of apoptosis

Chondrocyte apoptosis was measured by flow cytometry apoptosis detection kit (Annexin V-fluorescein isothiocyanate [FITC]/Propidium iodide [PI] kit, Beyotime). After re-suspending in Annexin V binding buffer, collected cells (0.5–1.0 × 105) were stained with FITC-labeled Annexin V solution in the dark at 4 ◦C for 15 min and then incubated with PI solution at 4 ◦C for 5 min. Stained cells were analyzed on a flow cytometry (BD Biosciences, San Jose, CA, USA). The percentages of IV gated cells were indicated as the early apoptotic cells.

2.5. Calcein-AM/PI double-staining

Calcein-AM/PI double-staining was used to evaluate apoptotic cell death. Collected cells (1.0 × 105/mL) were stained by a mixture of calcein-AM (2 mM) and PI solution (4.5 mM) (Yeasen, Shanghai, China) at 37 ◦C for 15 min. Apoptotic cell death (red) were examined under an inverted fluorescent microscope (Nikon, Japan). The percentages of cell death were evaluated from five random fields by calculating the number of PI-stained (dead, red) and calcein-AM-stained (live, green) cells.

2.6. Western blotting analysis

Protein was extracted from cultured cells with radioimmuno- precipitation assay buffer with freshly added 0.01% protease inhibitor cocktail (Solarbio, Beijing, China). Equal amounts of protein were separated by 10% or 15% SDS-PAGE, and transferred onto a nitrocellulose membrane (Millipore, Bredford, USA). The protein levels were analyzed by western blotting with specific antibodies. Antibodies against Bcl-2 and Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PRDX4 antibody was from Abcam. Antibodies against phospho-AKT (Ser 473), AKT, phospho-PRAS40 (Thr 246), PRAS40 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase-conjugated goat anti-mouse and anti- rabbit IgG antibodies were purchased from Beyotime. The blots were incubated with an enhanced chemiluminescence substrate mixture (Millipore). The densities of the protein bands were quantified using Image J (, Bethesda, MD, USA). Blots were normalized with signals from GAPDH.

2.7. Measurement of intracellular reactive oxygen species (ROS)

Generation of intracellular radicals and superoxide anion (O2—) were measured using dichlorodihydrofluorescein diacetate (DCFH- DA, Beyotime, Shanghai, China) and dihydroethidium (DHE) staining (Vigorous Biotech. Beijing, China) following the manu- factures’ instruction, respectively. Briefly, chondrocytes were harvest and incubated with 10 mM DCFH-DA or 50 mM DHE in the dark at 37 ◦C for 20 min. The cells were then rinsed with DMEM, and analyzed on a flow cytometry (BD Biosciences) with excitation wavelength of 480 nm and emission wavelength of 525 nm. ROS production in the experimental groups was normalized to the Control group.

Fig. 1. Identification of primary cultured chondrocytes. Isolated chondrocytes were stained with rabbit anti-Collagen II or anti-SOX9 as an indication of functional chondrocytes. For control staining, normal rabbit IgG was used as primary antibody. Magnification: 200×.

2.8. NO production

NO concentrations in the medium were determined by the Griess reaction method. Briefly, cultured medium were collected from treated cells, mixed with equal volume of Griess reagent (Jiancheng Bioengineering Institute, Nanjing, China), and incubat- ed in the dark for 10 min. The absorbance was measured at 550 nm on a microplate reader (Bio-Rad; Richmond, CA, USA).

2.9. Caspase-3 and Caspase-9 activity assay

Caspase-3 and -9 activity was determined with Caspase colorimetric assay kit (Kengen Biotech., Nanjing, China) per the manufacturer’s protocol. In brief, collected cells were lysed in the provided lysis buffer. Absorbance values were read at 400 nm on a microplate reader (Bio-Rad). Caspase activity in the experimental groups was normalized to the Control group.

Fig. 2. Protective effect of PRDX4 on IL-1b-induced chondrocyte apoptosis. (A) Western blotting was performed to assess PRDX4 levels. (B, C) Chondrocytes were infected with Vector or PRDX4 expressing virus. After 24 h, cells were treated with or without AZD5363 for 2 h prior to 24-h treatment with 10 ng/mL IL-1b. Annexin V/PI staining followed by flow cytometry assays (B) and Calcein-AM/PI double staining followed by observation with a fluorescence microscope (C) were conducted to evaluated chondrocyte apoptosis. All experiments were performed in triplicated and repeated for three times. All data are shown as mean SD. ***P < 0.001 versus Control; ###P < 0.001 versus IL-1b+Vector; ++P < 0.01 versus IL-1b+PRDX4. Fig. 3. Effects of PRDX4 and IL-1b on AKT signaling. (A) Chondrocytes were infected with Vector or PRDX4 expressing virus. After 24 h, cells were treated with or without AZD5363 for 2 h prior to 24-h treatment with 10 ng/mL IL-1b. Chondrocytes treated with 10 ng/mL IGF-1 (Peprotech.) for 24 h were served as positive control. Western blotting analysis of p-AKT and AKT. GAPDH was served as loading control. ***P < 0.001 versus Control; & P < 0.05 versus IL-1b. ##P < 0.01 versus IL-1b+Vector; +P < 0.05 versus IL-1b+PRDX4. (B)The chondrocytes were treated as described above. Western blotting was performed to assess the levels of p-PRAS40 and PRAS40. All data are shown as mean SD. ***P < 0.001 versus Control; ###P < 0.001 versus IL-1b+Vector; ++P < 0.01 versus IL-1b+PRDX4. 2.10. Statistical analyses All experiments were performed in triplicated and repeated at least three times and data are expressed as the mean SD. Comparisons were made by using by ANOVA test. Statistical significance was defined as P < 0.05. 3. Results 3.1. Effects of PRDX4 overexpression on IL-1b-induced chondrocyte apoptosis via PI3K/AKT signaling Chondrocytes were isolated from rat knee joint and immuno- histochemistry (IHC) stained with antibody against collagen II [26] or SOX9 [27], two well-known markers of chondrocytes. The results demonstrated that primary chondrocytes have been isolated and could be used for follow assays (Fig. 1). To study the effects of PRDX4 in the biological behavior of chondrocytes, the chondrocytes were treated with IL-1b (10 ng/mL) and PRDX4 expressing or Vector lentivirus. As shown in Fig. 2A, PRDX4 expression was significantly increased by PRDX4 overexpression (P < 0.001). We then assessed the apoptosis of chondrocytes by flow cytometry analysis following Annexin V/PI staining (Fig. 2B). The results showed that the percentage of cells undergoing early apoptosis was significantly increased after 24 h- exposure of IL-1b compared to that in the control group (P < 0.001). Infection with PRDX4 expressing virus significantly decreased the percentage of IL-1b-induced apoptotic chondro- cytes (P < 0.001). Apoptotic cell death was also detected by inverted fluorescence microscope after Calcein-AM/PI double-staining. Similar results were obtained (Fig. 2C). Fig. 4. PRDX4 reduces ROS and NO production. The chondrocytes were treated as described above. (A) The levels of intracellular radicals were measured by DCFH-DA labeling and flow cytometry analysis. (B) The levels of superoxide anion (O2—) were measured by DHE staining and flow cytometry analysis. (C) The concentration of NO in the medium was measured by the Griess reaction method. All experiments were performed in triplicated and repeated for three times. All data are shown as mean SD. ***P < 0.001 versus Control; ### P < 0.001 versus IL-1b+Vector; ++P < 0.01 and +++P < 0.001 versus IL-1b+PRDX4. To further explore the involvement of PI3K/AKT signaling in PRDX4 protection role, the AKT-specific inhibitor AZD5363 was added together with PRDX4 overexpression. As shown in Fig. 2B and C, AZD5363 reduced the suppression effects of PRDX4 on chondrocyte apoptosis induced by IL-1b (P < 0.01). Western blot analyses were then performed to study the changes of AKT activation (Fig. 3A). IGF-1 treatment, served as a positive control [28], caused an increase in AKT phosphorylation as compare to the basal levels (P < 0.001). Treatment with IL-1b, AZD5363 or IL-1b plus AZD5363 significantly decreased the levels of p-AKT compared to the control group (P < 0.001). Notably, PRDX4 overexpression significantly increased p-AKT in IL-1b- treated chondrocytes (P < 0.01). Additional AZD5363 treatment blocked the effects of PRDX4 overexpression on p-AKT and p- PRAS40 (P < 0.05). Total levels of AKT remained unchanged under all treatment. The phosphorylated levels of PRAS40, a downstream marker of AKT activity[29], was also changes with the alterations of p-AKT in chondrocytes (Fig. 3B). These results suggested that the protective effects of PRDX4 involved PI3K/AKT signaling. 3.2. Effect of PRDX4 on IL-1b-induced ROS and NO production Exposure of chondrocytes to IL-1b could increase ROS production and NO release. We also explored the changes of ROS and NO production (Fig. 4). DCF-DA and DHE staining were conducted to detect intracellula radicals and superoxide anion (O2—), respectively. NO production was assessed by Griess reaction method. IL-1b (10 ng/mL) could significantly induce ROS and NO production (P < 0.001). PRDX4 overexpression attenuated such effects (P < 0.001), while AZD5363 blocked the effects of PRDX4 overexpression (P < 0.01). 3.3. Effects of PRDX4 overexpression on IL-1b-induced mitochondrial apoptosis pathway and Caspase-3/9 activation To explore whether PRDX4 affected the mitochondrial apopto- sis pathway, protein levels of anti-apoptotic protein Bcl-2 and pro- apoptotic protein Bax were detected by Western blotting analysis. As illustrated in Fig. 5A, IL-1b significantly reduced Bcl-2 expression but induced Bax expression compared to the control group (P < 0.001), while such effects were partially reversed by PRDX4 overexpression (P < 0.001). Cells pretreated with PRDX4 and AZD5363 before IL-1b exposure had similar protein levels of Bcl-2 and Bax as cells treated with IL-1b only. The activation of Caspase-3/9 was also assessed. As shown in Fig. 5B, PRDX4 overexpression significantly inhibited IL-1b- induced Caspase-3/9 activation (P < 0.001). This effect was partly weakened by AZD5363 (P < 0.01), suggesting that PRDX4 exerted its effects via activating PI3K/AKT signaling. 4. Discussion Despite the extensive research efforts of the past decades, we still have little knowledge about the pathological mechanisms of OA. Emerging evidence has suggested that ROS plays a critical role in the initiation and progression of OA by inducing chondrocyte apoptosis and matrix degradation [5–8]. RDX4 is a member of antioxidant family proteins, which can reduce the intracellular ROS levels [19]. Increased IL-1b has implicated in OA pathophysiology. In the current study, to determine whether PRDX4 had protective effects on chondrocytes, chondrocytes were ectopic expressed with PRDX4 and then stimulated with IL-1b to emulate the damage in OA. The results indicated that PRDX4 overexpression significantly attenuated IL-1b-induced apoptosis. Bcl-2 family proteins can regulate mitochondrial outer membrane permeabi- lization, and activate Caspases to initiate apoptosis [30]. Presently, our data demonstrated that PRDX4 overexpression alleviated the effects of IL-1b that increased the ratio of Bax to Bcl-2 and Caspase activity. Thus, we supposed that the regulation of PRDX4 on chondrocyte apoptosis was via the mitochondrial apoptosis pathway. Studies have linked the endogenous ROS generation to mitochondrial apoptosis [31]. We also observed that the level of ROS and NO in IL-1b-treated chondrocytes with PRDX4 over- expression was lower than that in chondrocytes treated with IL-1b only. Therefore, we deduced that IL-1b treatment significantly induced ROS production and NO release, and then increased mitochondrial apoptosis, while PRDX4 overexpression can induce ROS scavenging and decrease NO release, thus attenuating cell apoptosis. Fig. 5. Protective effect of PRDX4 on IL-1b-induced chondrocyte apoptosis via inhibiting mitochondrial apoptosis pathway and Caspase-3/9 activation. The chondrocytes were treated as described above. (A) Western blotting was performed to assess Bcl-2 and Bax levels. (B) The activity of Caspase-3 and Caspase-9 was determined using Caspase colorimetric assay kit. All experiments were performed in triplicated and repeated for three times. All data are shown as mean SD. ***P < 0.001 versus Control; ###P < 0.001 versus IL-1b+Vector; +P < 0.05 and + +P < 0.01 versus IL-1b+PRDX4. As is known, PI3K/AKT pathway actively participate in the regulation of cellular growth and cell survival (anti-apoptosis) [32,33]. It inhibits cell apoptosis by regulating Bcl-2 family protein expression [33]. The present study showed that a 24 h-treatment with IL-1b significantly suppress AKT activity in chondrocytes, which was in line with previous findings [14,16]. Although phosphorylation events are generally considered rapid, transient events occurring within minutes, the suppression of AKT phosphorylation may last for a long period. Moreover, PRDX4 overexpression was able to alleviate the inhibitory effects of IL-1b on AKT activity in chondrocytes. More importantly, ectopic expression of PRDX4 in IL-1b-treated chondrocyte enhanced Bcl-2 expression, inhibited Bax expression, decreased Caspase-3/9 activation, and reduced ROS and NO production. The involvement of PI3K/AKT signaling was indicated by the experiments with AKT inhibitor AZD5363. The subcellular location of PRDXs has important implications for their function. Previous studies have showed that PRDX4 is localized in cytoplasm, Golgi and endoplasmic reticulum [34–37]. By immunofluorescence staining, we found that PRDX4 was localized in endoplasmic reticulum and the cytosol of rat chondrocytes (Fig. S1), thus it is reasonable that PRDX4 could exerting its effects on cytoplasmic protein AKT.

In conclusion, our findings demonstrated that the antioxidant enzyme PRDX4 inhibited IL-1b-induced chondrocyte apoptosis via scavenging of intracellular ROS and decreasing NO release. These effects involved in PI3K/AKT signaling pathway although the detailed mechanisms were not clear. Collectively, these results indicate the potential protective role of PRDX4 in OA cartilage degeneration. To validate this, it will be essential to test the protective effects of PRDX4 in animal models.

Competing interests

The authors declare that they have no competing interests.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at biopha.2017.03.075.


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