Human articular cartilage samples from 11 patients with OA (range, 61–72 years) were obtained at the time of total knee replacement. Normal cartilage was obtained from eight post-mortem donors (range, 61–69 years) with no previous history of joint pain. Informed consent was obtained in accordance with the local ethics commission and all studies were conducted under the approval of the Research Ethics Board of The Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Cartilage samples were harvested and fragmented into small pieces. Following the digestion of the cartilage samples with collagenase D, the resulting cells were incubated at 37°C in a 5% CO₂ humidified atmosphere in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

Articular chondrocytes were isolated from the femoral heads, femoral condyles and tibial plateau of 5- to 6-day-old C57BL/6 mice. Briefly, articular cartilage tissues were minced (<1 mm³), and digested with 0.3 and 0.05% collagenase D at 37°C for 45 min and overnight, respectively. The cell suspension was filtered through a sterile 40 μM nylon mesh cell strainer and the released cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified 5% CO₂ atmosphere. Cell viability of isolated chondrocytes was determined by trypan blue exclusion assay. Primary cells were maintained as a monolayer culture throughout this study.

Apoptotic cells were detected by fluorescence microscopy with cell permeable Hoechst 33258 dye (Invitrogen Life Technologies, Carlsbad, CA, USA). Following 48 h of transfection with siRNA fragments (Shanghai Genepharma, Co., Ltd, Shanghai, China), a subconfluent monolayer of mouse chondrocytes was subjected to stress with 10 ng/ml IL-1β for 48 h and stained with Hoechst 33258 dye (5 μg/ml). The nuclear morphology of cells was examined for the presence of condensed nuclei (apoptotic cells) and images were captured on an inverted fluorescence microscope (Axio Observer Z1; Carl Zeiss AD, Germany). The percentage of apoptotic cells was calculated from a minimum of 8 fields with >100 cells/field in an unbiased manner and the cells were scored in a blinded manner without previous knowledge of their treatments.

Immunohistochemistry for PUMA was performed using a polymer-based technology (Envision; Dako, Glostrup, Denmark). Briefly, human articular cartilage samples were dissected and post-fixed overnight in freshly prepared 4% paraformaldehyde in PBS (pH 7.4) at 4°C. Subsequently, the tissue samples were embedded in paraffin, cut into 5 μm thick sections and mounted onto gelatin-coated slides. Paraffin sections were incubated for 1 h at 60°C, deparaffinized in xylenes and rehydrated in a series of graded alcohols. Following three washes in phosphate-buffered saline (PBS; pH 7.4), endogenous peroxidase was inactivated by incubation in 3% H₂O₂ in methanol for 10 min. Antigen retrieval was then performed by microwaving slides in 10 mM sodium citrate buffer (pH 6.0) for 15 min followed by cooling to room temperature. Sections were incubated with an anti-PUMA antibody (1:100) (ab33906; Abcam, Cambridge, MA, USA) at 4°C overnight. Following washing in PBS, a polymer reagent was applied for 30 min and the expression of PUMA was visualized by incubation with diaminobenzidine tetrahydrochloride (DAB). Sections were then washed with PBS, counterstained with hematoxylin for 1 min, dehydrated for 15 min and mounted in neutral gum. The expression of PUMA was evaluated by IPP (version 6.0; Media Cybernetics, Silver Spring, MD, USA) following the collection of ten digital images at 1360×1024 pixel resolution at a magnification of ×400 using a BX51WI microscope (Olympus, Tokyo, Japan). The measurement parameters included density mean, area sum and inter ocular distance (IOD). Following calibration of the optical density, the image was converted to gray scale and the values were counted.

Whole cell lysates were prepared from chondrocytes lysed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mM NaF, 0.1 mM Na₃VO₄ and 1 mM phenylmethylsulfonyl fluoride). Lysates were cleared by centrifugation at 30,000 × g for 10 min at 4°C and protein concentrations were determined by an Bradford assay. A 30 μg aliquot of each cell lysate was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as described by 22506026. Proteins were then transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA) and membranes were probed with polyclonal antibodies specific for PUMA (diluted 1:1,000; Abcam), Bax, c-Jun and GAPDH (diluted 1:1,000; Cell Signaling Technology, Inc) overnight at 4°C. Following washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:1,000; Jackson ImmunoResearch, West Grove, PA, USA) for 60 min and the proteins were visualized using the ECL chemiluminescence system (Forevergen Bioscience, China).

Total RNA was extracted from isolated chondrocytes using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s recommendations. First strand cDNA was synthesized from 1 μg of total RNA using M-MLV Reverse Transcriptase (Promega, Southampton, UK) and oligo(dB) primers. Reverse transcription was performed using SuperScript reverse transcriptase (Invitrogen Life Technologies) and oligo-dT primers. The following sense and anti-sense primers for Puma, c-jun, and β-actin were used: Puma: forward, 5′-AGCGGCGGAGACAAGAA-3′ and reverse, 5′-CAAGTCCGTATCTCCATCAGTG-3′; c-jun: forward, 5′-CCTTCTACGACGATGCCCTC-3′ and reverse, 5′-GGTTCAAGGTCATGCTCTGTTT-3′; β-actin: forward, 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse 5′-CCAGTTGGTAACAATGCCATGT-3′.

Mouse chondrocytes were transfected with 100 nM of siRNA against Puma using Lipofectamine reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. siRNA sequences were as follows: si-PUMA-1, 5′-CCTGGAGGGTCATGTACAATCTCTT-3′; si-PUMA-2, 5′-GGAGGGTCATGTACAATCTCTTCAT-3. One scrambled siRNA was designed to serve as a siRNA transfection control with the following sequence; 5′-UGGUUUACAUGUCGACUAA-3′. The cells were lysed 72 h following siRNA transfection and the degree of knockdown was assessed by RT-PCR and western blot analysis.

The apoptosis of chondrocytes has a key role in the development of OA, however, the mechanisms responsible for the induction of apoptosis remain unclear. In order to study the apoptotic signaling pathway, mouse chondrocytes were cultured and treated with 10 ng/ml of IL-1β to mimic the pathogenesis of OA. Since it has been demonstrated that PUMA regulates extracellular apoptotic pathways, we investigated the effects of IL-1β treatment on PUMA levels during chondrocyte apoptosis. As illustrated in Fig. 1A, chondrocytes underwent apoptosis after 48 h of treatment with IL-1β. The apoptotic rate was quantified following Hoechst staining and demonstrated that to be ~23% in treated chondrocytes, however only 3% in untreated cells (Fig. 1B). To determine PUMA protein and mRNA levels, IL-1β-treated chondrocytes were then subjected to western blotting and RT-PCR. As illustrated in Fig. 1C, the PUMA protein and mRNA levels were significantly increased throughout the time course of IL-1β treatment. Together, these data indicate that IL-1β treatment not only has the ability to induce chondrocyte apoptosis, but also to alter PUMA protein and mRNA levels during the apoptotic process.

Since PUMA mRNA and protein levels were upregulated in cultured chondrocytes following IL-1β stimulation, we then investigated whether PUMA upregulation was essential for chondrocyte apoptosis. Two different siRNA fragments specifically targeting PUMA were designed. The knockdown efficiency of each siRNA was determined by RT-PCR and western blot analysis following 72 h of transfection. Of the two siRNAs tested, si-PUMA-2 gave an enhanced knockdown efficiency at a concentration of 100 nM. As demonstrated in Fig. 2A (upper panel), the mRNA of PUMA was decreased by si-PUMA-1 and si-PUMA-2, respectively, as compared with the scrambled siRNA. PUMA protein levels were also significantly reduced upon treatment with si-PUMA-1 and si-PUMA-2 (Fig. 2A, lower panel), indicating a successful knockdown effect with these siRNA fragments. In addition to this, knockdown of PUMA triggered a marked decreased in the apoptotic rate in the presence of IL-1β (Fig. 2B). These data suggest that PUMA is critical for IL-1β induced chondrocyte apoptosis.

Since the JNK/c-Jun pathway has been reported to regulate PUMA induction, it was considered that this pathway may also be involved in the induction of PUMA in an IL-1β-induced model of apoptosis in mouse chondrocytes. Two potent and specific inhibitors of the JNK/c-Jun pathway were used; SP600125 to inhibit JNK and CEP11004 to inhibit MLK, an upstream kinase of JNK. The results identified an increased phosphorylation of JNK and c-Jun following IL-1β treatment, indicating activation of the JNK/c-Jun pathway (Fig. 3A). Upon the addition of 10 μM SP600125 or 2 μM CEP11004, there was a decrease in the phosphorylation levels of c-Jun and JNK, as well as a decrease in the protein levels of PUMA (Fig. 3A). PUMA protein levels were decreased with the two inhibitors following IL-1β treatment (Fig. 3A). Simultaneously, PUMA mRNA was also suppressed upon treatment with SP600125 or CEP11004 in the presence of IL-1β (Fig. 3B).

Following this, we determined whether inhibition of the JNK/c-Jun pathway would impact the rate of apoptosis induced by IL-1β. As demonstrated in Fig. 3C, apoptosis induced by IL-1β was significantly suppressed following the incubation of cells with SP600125 and CEP11004, respectively. This accumulative evidence suggests that the JNK/c-Jun pathway may be activated by IL-1β treatment and that it acts upstream of PUMA to mediate chondrocyte apoptosis.

To determine whether c-Jun and PUMA are upregulated in chondrocyte tissues isolated from OA patients, the expression level of c-Jun and PUMA was examined in human articular cartilage and in primary cultured chondrocytes from OA patients. Using immunohistochemistry, an increased staining of PUMA was observed in the articular cartilage of OA patients as compared with the normal tissues (Fig. 4A). Positive staining for PUMA was identified in 56% of OA tissues tested (data not presented). Then, chondrocytes from the tissues of patients with or without a history of OA were isolated and cultured. Cell lysates were prepared and subject to western blot analysis. As compared with chondrocytes derived from healthy tissue (Fig. 4; NC1,2,3), the protein levels of c-Jun and PUMA in chondrocytes from OA patients were elevated (Fig. 4; OA1, 2, 3). Similarly, the mRNA levels for c-Jun and PUMA were also elevated in OA patients (Fig. 4, lower panel). These data demonstrated that PUMA and c-Jun are significantly upregulated in OA patients, suggesting they have a role in the apoptosis observed in the tissues of OA patients.