The expression of p-ATF2 involved in the chondeocytes apoptosis of an endemic osteoarthritis, Kashin-Beck disease

Specimens of human articular cartilage were collected from a total of 15 KBD patients (9 males and 6 females, 44-58 years of age) who were diagnosed as the second or third degree of KBD, based on the Diagnosing Criteria to Kashin-Beck disease in China [15, 22]. The donors were inhabitants of endemic regions of Linyou, Yongshou, and Qianyang in Shaanxi Province of China. Normal human knee cartilage samples were collected from donors (4 males and 2 females, 38-55 years-of age) who were suffering from traffic accidents and undergoing total knee replacement surgery from non-KBD areas. The health status of control cartilage samples was examined through histological examination of hematoxylin-eosin stained sections to exclude osteoarthritis (OA), rheumatoid arthritis (RA) and other bone and cartilage diseases. Permission for this study was given by the Human Ethics Committee of the Xi’an Jiaotong University. All patients and normal donors provided a written informed consent for participation in the study and publication their individual clinical details and clinical images.

Cartilage tissues of every KBD donor were divided into two parts, so that one part was used to extract mRNAs and proteins, while the other part was used for cell culture. One representative patient with grade III KBD, the radiographic images of the left knee of a patient with grade II KBD and HE staining pictures of articular cartilage from normal and KBD are shown in Figure 1. Meanwhile, proteins extracted from every normal donor were kept separately and the rest cartilage tissues of normal donors were used for cell culture.

Within 8 h after surgery, the cartilage tissue was collected and washed with phosphate-buffered saline (PBS) three times, then the cartilage was cut into small pieces, which were incubated at 37°C with trypsin for 10 min. After removing the trypsin solution, the cartilage was digested at 37°C with type II collagenase using 1 ml of digestion solution per 100 mg tissue (Gibco, Grand Island, NY, USA). Every 3 hours, the digested fluid was collected through gauze to remove undigested cartilage fragments, then we collected the chondrocytes by centrifugation and reused the collagenase solution to digest the cartilage pieces. This protocol was repeated up to three times until we got enough chondrocytes for further experiments. Chondrocytes from individual donors were kept strictly separated in all experiments. Cells were plated in cell culture flasks in DMEM/F12 (1:1) (HyClone, Thermo Scientific, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin, and maintained in a humidified atmosphere at 5% CO₂ and 37°C. The medium was renewed two or three times a week according to the cell growth state. Primary cells were used in all experiments.

Total RNAs were extracted using RNAfast200 kit (Fastagen, Shanghai, China), whose quality and concentration were assayed by a NanoDrop spectrophotometer (Thermo Scientific, USA), and then subjected to reverse transcription using RevertAidTM First Strand cDNA Synthesis kit (Fermentas, MBI, Vilnius, Lithuania) by Eppendorf gradient type mastercycler (Eppendorf, Hamburg, Germany). Reverse transcription products were used for quantitative real-time PCR analysis performed with iQTM5 Real-Time PCR Detection Systems device (Bio-Rad, Philadelphia, PA, USA) using BioEasy SYBR Green I Real Time PCR Kit (Bioer, Hangzhou, China) with oligonucleotide pairs specific for human JNK, p38, ATF2 and GAPDH with the following cycling conditions: 94°C for 2 mins, 94°C for 10 s, 58°C for 30 s, and 72°C for 30 s for 40 cycles, followed by a melting curve analysis. The forward and reverse primer pairs designed to generate a 102-bp fragment of JNK, a 107-bp fragment of p38, a 104-bp fragment of ATF2, and a 226-bp fragment of GAPDH (an internal control) are presented in Table 1. The results of relative gene expression data were analyzed using Real-Time quantitative PCR and the 2^-∆∆CT method [23].

The proteins from cartilage and chondrocytes of KBD patients and from normal human cartilage were extracted with RIPA Cell Lysis Solution (Beyotime, Jiangsu, China), which was supplemented with PMSF and PhosSTOP alkaline phosphatase inhibitors (Hoffmann-La Roche Ltd, Basel, Switzerland). Protein concentration was assayed to adjust an equivalent loading dose of 50 μg. Before electrophoresis, all the samples were boiled in sodium dodecylsulfate (SDS) sample buffer for 5 mins. The lysates were separated by polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was then incubated at room temperature in a blocking solution composed of 5% skimmed milk powder dissolved in Tris-buffered saline (TBS), pH 7.4, containing 0.1% Tween-20 for 1 h, followed by incubation in the blocking solution with anti-human JNK, p-JNK, p38, p-p38, ATF2, p-ATF2 antibodies separately at 4°C overnight. All the antibodies were purchased from Bioworld Technology (St. Louis Park, MN, USA; Cat No: BS3631, BS4763, BS3567, BS4766, BS1021, BS4018). The membrane was washed three times in TBS-Tween (5 mins each), and then incubated with an HRP-conjugated IgG antibody in the blocking solution. After washed three times in TBS-Tween and once in TBS (5 mins each), the immunoreactive protein was visualized using an enhanced chemiluminescence detection kit (ECL, Thermo Scientific, USA). To show equal loading of the protein samples, β-actin was used as internal control.

In order to understand if JNK and p38 activated in the apoptosis of KBD chondrocytes, we seeded primary KBD chondrocytes on 6-well dishes and divided into 3 groups: 1) control group, 2) p38 inhibitor (SB203580) group, 3) JNK inhibitor (SP600125) group. The effective inhibition doses of SB203580 for p38 and SP600125 for JNK protein with 10 μM and 20 μM were tested with KBD chondrocytes.

For control groups we follow the above mentioned chondrocytes culture protocol, while for JNK and p38 inhibitors groups, the culture medium was added with SB203580 and SP600125 individually before used for cell cultures. One week later, early chondrocyte apoptosis rates of the 3 groups were determined by a flow cytometer (Becton Dickinson, Mountain View, CA, USA) using AnnexinV-FITC Apoptosis Detection kit according to the manufacturers’ instructions (KenGEN, Nanjing, China). Moreover, apoptotic morphological changes in the nuclear chromatin were detected by DAPI stain. Chondrocytes were washed with PBS and incubation with DAPI stain solution for 10 min (Beyotime, Haimen, China). Then the chondrocytes were viewed under a fluorescence microscope (Olympus, IX-70, Japan) within 2 h. Furthermore, real-time quantitative PCR and western blot analyses were used to compare mRNA (NM_002750, NM_001315, NM_001880) and protein (p-JNK, p-p38, ATF2, p-ATF2) levels among the groups.

Gene expression profiles may vary according to cell culture conditions. Therefore, we performed a quantitative RT-PCR analysis to compare the levels of ATF2, JNK and p38 in KBD cartilage and those in cultured KBD chondrocytes from the same donors. To our surprise, it could be observed that the expression levels of JNK and ATF2 mRNAs were about 3.2-fold (p < 0.01) and 2.7-fold (p < 0.05) higher in cartilage samples than in chondrocytes, while p38 levels remained stable with 1.3-fold in cartilage compare with chondrocytes (Figure 2).

In order to find out whether the observed changes in mRNA levels were reflected at the protein levels, western blot analysis was performed. The protein levels of p38, p-p38 and JNK were detected with a significant increase in KBD cartilage compared with KBD chondrocytes (p < 0.05, and were 0.025, 0.040 and 0.032 respectively). Interestingly, the p-JNK, ATF2 and p-ATF2 could only be detected in KBD cartilage samples; the decrease in JNK and ATF2 total protein levels in cultured chondrocytes was in line with the lower level of their mRNA expression respectively (Figure 3A).

Whether the expression of p-p38, p-JNK, ATF2, and p-ATF2 was specific to KBD was unknown, western blot analysis of samples isolated from three normal donors and three KBD patients was performed. It could be observed that only the phosphorylated ATF2 could be detected in the cartilage of KBD patients, while the expression of p-p38, p-JNK (p = 0.015) and ATF2 (p = 0.005) increased dramatically in the KBD cartilage when compared with that in normal cartilage (Figure 3B).

Western blot analysis of cartilage and chondrocyte samples isolated from three normal donors was performed. The level of p-p38 in normal cartilage was a bit higher than that in normal chondrocytes. Meanwhile, the levels of ATF2 (p = 0.059) and phosphorylated JNK (p = 0.032) were detected in the cartilage samples other than in the chondrocyte samples, and phosphorylated ATF2 was absent in both cartilage and chondrocyte samples (Figure 3C).

Figure 4 showed that 10 μM and 20 μM for each inhibitor showed targeted phosphate protein blocked. Therefore, lower dose with 10 μM was selected as the effective inhibition dose of SB203580 for p38 and SP600125 for JNK.

In control group, the early apoptosis rate was 7.2 ± 1.3% (Figure 5A). The JNK inhibitors SP600125 decreased apoptosis rate from 7.2 ± 1.3% to 3.4 ± 1.1% (p < 0.05), while p38 inhibitor SB203580 had a less pronounced effect on the percentage of apoptosis rate (5.8 ± 1.4%).

As shown in Figure 5B, DAPI-stained nuclei from chondrocytes of control group showed cytoplasmic signs of apoptotic cell death, meanwhile, the nuclear fragmentation and condensation were evident. In contrast, KBD nuclei were bigger and rounder with inhibitors.

The expression levels of the p38, JNK and ATF2 mRNA were higher in the control group (Figure 5C). The addition of SP600125 reduced the expression of JNK and ATF2 mRNAs to less than 0.5-fold each (p < 0.05); SB203580 decreased the level of p38 mRNAs to about 0.5-fold as well while had a less effect on the level of ATF2 mRNA to 0.8-fold.

The p38 inhibitor SB203080 specifically prevented phosphorylation of p38 (Figure 5D), while affected slightly to p-JNK, ATF2 and p-ATF2; meanwhile, the JNK inhibitor SP600125 efficiently blocked the phosphorylation of JNK and the total protein level of ATF2 as well as p-ATF2. SP600125 was not only a potent inhibitor of ATF2 phosphorylation; it also decreased the protein amount of ATF2, which was in line with its mRNA expression.