Two-month-old male Norway rats (280–300g; n=42) were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China). All rats were randomly assigned to two groups: sham surgery group (n=14) and OA model group (n=28). All animal studies were conducted according to the guidelines of animal care and were approved by the Animal Care Committee of Shanghai Jiao Tong University.
The following reagents were used: TGF-β1 (10 ng/ml; R&D Systems, Minneapolis, MN), MG132 (5 µM; Sigma-Aldrich, St. Louis, USA), Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific, Waltham, USA), cycloheximide (100 μg/ml; Sigma-Aldrich), PD98059 (10 µM; Sigma-Aldrich).
In vivo rat OA model
Twenty-four 2-month-old male Norway rats were anaesthetized by intraperitoneal injection with 3% pentobarbital sodium until cessation of all sensory reflexes. The right knees of rats were routinely disinfected with povidone iodine before surgery. The right knee joint was exposed by incision from the medial side of the patella, and the medial collateral ligament was removed to open the articular cavity. The rats then underwent anterior cruciate ligament transection in combination with partial medial meniscectomy (ACLT+pMMx) on the right knee as described previously [17, 18]. Finally, the incisions were sutured and ACLT+pMMx surgeries were induced after complete hemostasis was achieved. In the control group, the sham operations were performed on the model rats. After 8 weeks, OA models were successfully induced in rats that received the ACLT +pMMx surgeries.
Preparation of AAV vectors
Adeno-associated virus (AAV) serotype 2 vectors are AAV plasmids that can encode target genes and enhanced green fluorescent protein (EGFP) cDNA under control of the cytomegalovirus (CMV) enhancer. According to the previously reported method , serotype 2 of AAV (AAV-CMV-rUSP15-FLAG-T2A-EGFP and pAAV-U6-rERK2 [shRNA-1]-EGFP) packaged with two plasmids (pAAV-RC and pAAV-Helper) was obtained. The AAV knockdown sequence targeting ERK2 used was 5′- CTTCCAACCTCCTGCTGAA-3′.
AAV intraarticular injections
The two sub-control groups that received the sham surgeries were detailed as follows: (1) 7 rats without any injection. (2) 7 rats received a 30 μL intraarticular injection of pAAV-U6-rERK2 [shRNA-1]-EGFP (2.5 × 1010 vg) in the right knee joint.
The rat OA model groups that received the ACLT+pMMx surgeries were divided into four groups, as follows: (1) 14 OA model rats without any injection. (2) 7 OA model rats received a 30 μL intraarticular injection of pAAV-CMV-rUSP15-FLAG-T2A-EGFP (2.5 × 1010 vg) in the right knee joint. (3) 7 rats received a 30 µL injection of pAAV-CMV-rUSP15-FLAG-T2A-EGFP (2.5 × 1010 vg) followed by another 30 µL injection of pAAV-U6-rERK2 [shRNA-1]-EGFP (2.5 × 1010 vg).
Immunohistochemistry, HE/Safranin-O fast green staining and histology
Knee joints were fixed in 10% neutral buffered formalin for 3 d and decalcified in 10% EDTA solution at 4 °C for 3 months. Samples were dehydrated in ethanol, embedded in paraffin, and cut into coronal sections. Immunohistochemical results were detected with the following antibodies: Aggrecan (ab3778, Abcam), Col2a1 (ab34712, Abcam), USP15 (ab71713, Abcam), ERK2 (sc-1647, Santa Cruz Biotechnology, Dallas, USA), p-SMAD2 (ab188334, Abcam), p-ERK1/2 (4370S Cell Signaling Technology, Danvers, USA). Cartilage was stained with HE/Safranin-O fast green staining to observe general morphological changes. And then hyaline cartilage (HC) thickness was counted by at least three participants. Cartilage histological properties were assessed by the Osteoarthritis Research Society International (OARSI) scoring system as previously described [20, 21]. Double-blind histological evaluations of the cartilage lesions were performed in the whole analysis process.
Cell culture, transient transfections, and lentiviral infections
Primary chondrocytes were isolated from rat cartilage tissue as previously described . Briefly, rats were euthanized by cervical dislocation and the hyaline cartilage of the femoral and tibial surfaces was removed with a scalpel. The pared hyaline cartilage was digested after cutting into pieces with hyaluronidase and collagenase II treatment. The isolated cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C under a humidified atmosphere of 5% CO2. To amplify the cultured cells, they were subcultured using 0.25% trypsin/EDTA. The isolated chondrocytes were identified by immunostaining for collagen II and Aggrecan.
ATDC5 cells (Riken Cell Bank, Tsukuba, Japan) were obtained and maintained in DMEM/F12 (1:1) (GIBCO) containing 5% FBS (HyClone, GE Healthcare Life Sciences, Chicago, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. To induce chondrogenic differentiation, medium was supplemented with 10 µg/mL of insulin-transferrin-selenium (Thermo Fisher Scientific) after confluence and cells were cultured for 14 days. 293T cells (Riken Cell Bank, Tsukuba, Japan) were cultured in high glucose DMEM supplemented with 10% FBS and D-glutamate.
Transient transfections and lentiviral infections were performed as previously described . HEK293T cells were transiently transfected with pLenti-CMV-mUSP15-FLAG-GFP-Puro, pLenti-CMV-mUSP15C269S-FLAG-GFP-Puro and pLenti-CMV-mERK2-HA-Puro. PLenti-CMV-mUSP15-FLAG-GFP-Puro, pLenti-CMV-mUSP15C269S-FLAG-GFP-Puro, pLenti-CMV-mERK2-HA-Puro, lentiCRISPRv2-USP15a, lentiCRISPRv2-USP15b, lentiCRISPRv2-ERK2a, lentiCRISPRv2-ERK2b and pLenti-CMV-Ub-His-Puro were co-transfected into HEK293T cells with the packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260). The relevant CRISPR sequence were as follows: USP15-a-F: 5′-CACCG GGTATCTAGTAGATAGTCGG-3′, USP15-a-R: 5′-AAAC CCGACTATCTACTAGATACCC-3′, USP15-b-F: 5′-CACCG TGGCGACGCGCAGTCACTTA-3′, USP15-b-R: 5′-AAAC TAAGTGACTGCGCGTCGCCAC-3′, ERK2-a-F: 5′-CACCG CAGAGTACGTAGCCACACGT-3′, ERK2-a-R: 5′-AAAC ACGTGTGGCTACGTACTCTGC-3′, ERK2-b-F: 5′-CACCG GGATATACTTTAGCCCTCTC-3′, ERK2-b-R: 5′-AAAC GAGAGGGCTAAAGTATATCCC-3′. ATDC5 cells were infected with the resulting stable lentiviral vectors.
Quantitative real-time RT-PCR
Total RNA was isolated from the ATDC5 cells or articular cartilage in rats by using Trizol (TaKaRa, Japan) according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA with Reverse Transcription Kit (TaKaRa, Japan). Relative mRNA amount was quantified by Light Cycler 480 system (Roche, Switzerland) with SYBR Green I Kit (TaKaRa, Japan). The mouse primer sequences of the Col2a1, Col10a1, Sox9, Runx2 and GAPDH used were as follows: Col2a1: 5’-TACTGGAGTGACTGGTCCTAAG-3’ (Forward) and 5’-AACACCTTTGGGACCATCTTTT-3’ (Reverse), Col10a1: 5’-GAATTTCTGTGCCAGGAAAACC-3’ (Forward) and 5’-TTTTCACCTCTTCTTCCCACTC-3’ (Reverse), Sox9: 5’-GAGTTTGACCAATACTTGCCAC-3’ (Forward) and 5’-GTAACTGCCAGTGTAGGTGAC-3’ (Reverse), Runx2: 5’-CCTTCAAGGTTGTAGCCCTC-3’ (Forward) and 5’-GGAGTAGTTCTCATCATTCCCG-3’ (Reverse), GAPDH: 5’-CACTCTTCCACCTTCGATGC-3’ (Forward) and 5’-TCTTGCTCAGTGTCCTTGCT-3’ (Reverse). The rat primer sequences of the Col2a1, Aggrecan, Sox9 and GAPDH used were as follows: Col2a1: 5’-GGAGCAGCAAGAGCAAGGAGAAG-3’ (Forward) and 5’-GGAGCCCTCAGTGGACAGTAGAC-3’ (Reverse), Aggrecan: 5’-GCTACGACGCCATCTGCTACAC-3’ (Forward) and 5’- ATGTCCTCTTCACCACCCACTCC-3’ (Reverse), Sox9: 5’-TGGCAGAGGGTGGCAGACAG-3’ (Forward) and 5’-CGTTGGGCGGCAGGTATTGG-3’ (Reverse), GAPDH: 5’- ATGGTGAAGGTCGGTGTGAA-3’ (Forward) and 5’-CACCACCCTGTTGCTGTAGC-3’ (Reverse). Relative amounts of mRNA were standardized and calculated as previously described [24, 25].
Cell extracts were subjected to SDS-PAGE in 10% polyacrylamide gels, followed by blotting onto polyvinylidene difluoride membranes (Millipore, Bedford, USA). Primary antibodies included p-ERK1/2, FLAG, Ubiquitination, GAPDH (Cell Signaling Technology,); ERK2, HA, His (Santa Cruz Biotechnology); USP15, p-SMAD2, SMAD2, SMAD4 (Abcam). Corresponding species-specific secondary antibodies (anti-rabbit IgG HRP, anti-mouse IgG HRP) were used. GAPDH was used to normalize the results adjusting for control variations between individual experiments. The results were detected by a Western Chemiluminescent HRP Substrate kit (Millipore, Burlington, USA) and imaged using the FluorChem M system (Protein Simple, San Jose, USA).
Immunoprecipitation and deubiquitination assay
Monolayer cultured cells were added to ice cold RIPA buffer and centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was transferred to a fresh 1.5 mL conical centrifuge tube on ice with 5 µL of indicated antibodies and 20 µL of resuspended volume of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology), and then incubated overnight at 4 °C on a rocker platform. Immunoprecipitates were collected and the supernatant discarded. The pellet was washed 4× with 1.0 mL RIPA buffer. After the final wash, samples were boiled for 10 min and analyzed by Western blotting. To detect ERK2 deubiquitination, transient transfections and lentiviral stable infections were performed before immunoprecipitation.
Immunofluorescence staining was performed as previously described [26, 27]. In brief, cells were fixed with 4% formaldehyde and incubated with USP15, p-ERK1/2, HA, FLAG, Aggrecan, Col2a1 and Col10a1 antibodies. Immunofluorescence staining results were detected with the following antibodies: Col2a1 (ab34712, Abcam), Aggrecan (ab3778, Abcam), Col10a1 (ab49945, Abcam) and other antibodies were described above. Samples were then treated by a secondary antibody. Nuclear DNA was visualized by DAPI staining and viewed under an immunofluorescence confocal microscope (NIKON Eclipse Ti, Japan).