BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as osteoarthritis progression in mice

BMP5 is overexpressed in the chondrocytes from the knee articular cartilage of patients with OA and DMM-OA model mice

We investigated the potential role of BMP5 in OA by analyzing knee articular cartilage tissues samples and joint fluid from OA patients that underwent total knee arthroplasty surgery and healthy patients that underwent amputation due to trauma. Immunohistochemistry (IHC) and western blotting analyses showed that BMP5 protein levels were significantly higher in the knee articular cartilage tissues from OA patients compared to those from the healthy subjects (Figure 1A–1C, 1E). ELISA results showed that the levels of BMP5 were higher in the synovial fluid of OA patients compared to those from healthy subjects (Figure 1F).

Next, we analyzed the levels of BMP5 in knee joint sections from DMM-2W (2 weeks after DMM surgery), DMM-4W (4 weeks after DMM surgery), and sham groups of mice. IHC analysis showed that BMP5 protein levels were higher in the knee cartilage sections from the DMM-4W and DMM-2W group mice compared to the sham-operated mice; moreover, BMP5 protein levels were higher in the DMM-4W mice compared to the DMM-2W mice (Figure 1D, 1G). Furthermore, BMP5 expression was significantly higher in the knee cartilage tissues from aged mice compared to those from young mice (Supplementary Figure 1A, 1B). These findings suggest that BMP5 overexpression may be involved in chondrocyte dysfunction and knee articular cartilage-related OA.

BMP5 silencing delays OA progression in the DMM model mice

To determine the potential role of BMP5 in OA development, we injected lentiviral-si-BMP5 (LV-si-BMP5) and LV-si-NC at 7 and 14 days after DMM surgery. We confirmed lentiviral transduction efficiency in the murine articular cartilage tissues by RT-PCR at 2 weeks after intra-articular injections (Figure 2B). BMP5 mRNA and protein levels were significantly reduced in the chondrocytes from the DMM + LV-siBMP5 group mice at four weeks after intra-articular lentiviral injections compared to those from the DMM + LV-si-NC group (Figure 2B and Supplementary Figure 2A, 2B).

Histological staining analysis of the knee cartilage specimens from DMM + LV-siBMP5 and DMM + LV-si-NC group mice is shown in Figure 2A, and the results of the OARSI scoring is shown in Figure 2D. At 4 weeks after DMM surgery, we observed reduced hyaline cartilage/calcified cartilage (HC/CC) ratio and the proportions of chondrocytes in the knee joint cartilage tissue and induced cartilage degradation, severe cartilage erosion, synovitis, and thickening of the subchondral bone plate (Figure 2A). However, compared to the DMM + LV-siNC group, the knee joint cartilage tissues from the DMM + LV-siBMP5 group mice showed reduced cartilage degradation (Figure 2A), lower OARSI grades (Figure 2D), and increased subchondral bone plate (SBP) at 2 and 4 weeks after DMM surgery (Figure 2C, 2E). Both LV-siNC and LV-siBMP5 groups showed similar levels of synovitis at 2 weeks and 4 weeks post-DMM surgery (Figure 2C, 2E). These results suggest that BMP5 promotes OA progression in the DMM model mice.

BMP5 silencing reduces OA-related pathology in both in vivo and in vitro OA models by decreasing the expression of catabolic factors in the chondrocytes

Next, we investigated if BMP5 silencing affects the expression levels of matrix-degrading enzymes and aggrecanase in chondrocytes in order to determine the mechanisms through which BMP5 promotes cartilage destruction in OA. We transfected chondrocytes with multiple siRNAs against BMP5 (BMP5-siRNA1, BMP5-siRNA2, and BMP5-siRNA3) and observed that BMP5-siRNA1 significantly reduced BMP5 mRNA and protein levels in the chondrocytes compared to siRNA2-BMP5, siRNA3-BMP5 and si-NC (Supplementary Figure 3A, 3B). Hence we selected BMP5-siRNA1 for subsequent experiments.

Previous studies in mice demonstrated that pro-inflammatory cytokines (TNF-α and IL-1β), anabolic factors (type II collagen and SOX9), and catabolic factors (MMP13 and ADAMTS-5) played critical roles in OA progression [29]. Therefore, we analyzed these factors in the chondrocytes from both in vitro and in vitro OA models. IL-1β-treated BMP-silenced chondrocytes showed significant reduction in the mRNA and protein levels of ADAMTS5 and MMP13, but there is no change in SOX9 mRNA compared to the IL-1β control group chondrocytes (Figure 3A, 3B). Moreover, we observed significant reduction in MMP13 levels and increased expression of Col2a and TIMP2 in the chondrocytes from the LV-siBMP5 group mice compared to those from the LV-si-NC group mice (Figure 3C, 3D). These results suggest that BMP5 silencing in the knee articular cartilage chondrocytes reduces pathological changes related to OA in the DMM-induced model mice by decreasing the expression of matrix catabolic factors.

Silencing of BMP5 inhibits chondrocyte senescence in both in vivo and in vitro OA models

We then performed SA-β-gal staining assay to determine the role of BMP5 in OA-related chondrocyte senescence. IL-1β-treated BMP5 knockdown chondrocytes showed significantly lower SA-β-gal activity than the corresponding controls (Figure 4A).

Furthermore, we assessed the protein levels of cyclin-dependent kinase inhibitors, p16, p21, p53, and HMGB1, which play a role in DNA damage-induced senescence [10, 21]. Western blotting analysis showed that p16, p21, HMGB1, and p53 protein levels were significantly reduced in the IL-1β-treated BMP5-silenced chondrocytes compared to the corresponding controls (Figure 4B).

Next, we analyzed γH2AX staining to determine if BMP5 knockdown reduced premature senescence of the chondrocytes. We observed that the relative area of γH2AX staining was significantly lower in the IL-1β-treated BMP5-depleted chondrocytes compared to the IL-1β-treated control chondrocytes (Figure 4C). We also demonstrated that p16 and p21 protein levels were significantly lower in the knee joint cartilage tissues from the DMM+LV-siBMP5 group mice compared to those from the DDM+LV-siNC group mice (Figure 4E, 4F). These data implied that BMP5 silencing reduced premature senescence of the chondrocytes in both in vivo and in vitro OA models.

Depletion of BMP5 suppresses chondrocyte apoptosis in both in vivo and in vitro OA models

Chondrocyte apoptosis plays a significant role in articular cartilage destruction and matrix degradation in OA, an age-related degenerative human disease [16]. Therefore, we analyzed if BMP5 modulated the expression of anti-apoptotic or pro-apoptotic proteins in IL-1β-stimulated control or BMP5-silenced murine chondrocytes using western blotting, TUNEL staining, and flow cytometry assays. The numbers of TUNEL-positive cells were significantly reduced in the IL-1β-stimulated BMP5-silenced murine chondrocytes compared to the corresponding controls (Figure 5A, 5B). Furthermore, western blotting analysis showed that cleaved caspase-3 levels were significantly reduced and Bcl-2 levels were significantly increased in the IL-1β-stimulated BMP5-silenced murine chondrocytes compared to the corresponding controls (Figure 5C). Flow cytometry analysis confirmed that BMP-5 silencing reduced apoptosis in IL-1β-stimulated BMP5-silenced murine chondrocytes compared to the corresponding controls (Supplementary Figure 4A, 4B). The numbers of TUNEL-positive chondrocytes were significantly reduced in the LV-siBMP5 group compared to the LV-siNC group (Figure 5D, 5E). Conversely, BCl2 expression was significantly higher in the chondrocytes from the LV-siBMP5 group compared to those from the LV-siNC group (Figure 5D, 5E). These results demonstrate that BMP5 silencing reduced IL-1β-induced and DMM-induced chondrocyte apoptosis.

BMP5 regulates chondrocyte senescence via p38/ERK signaling pathway

Next, we investigated the molecular mechanisms underlying BMP5 that regulate OA progression in the articular cartilage tissues. ERK1/2 signaling pathway plays a crucial role in promoting cellular senescence [30]. Therefore, we analyzed the status of the p38/ERK signaling pathway in control and BMP5-silenced chondrocytes subjected to IL-1β treatment by western blotting. The levels of total and phosphorylated p38 and ERK1/2 proteins were significantly higher in the IL-1β-treated control chondrocytes compared to the IL-1β-treated BMP5-silenced chondrocytes (Figure 6A). Moreover, the levels of p38 and ERK1/2 proteins were significantly increased in a dose-dependent manner by incubating chondrocytes with recombinant BMP5(Rocky Hill, NJ, USA, 120-39.), rhBMP5 (Figure 6B). Furthermore, IL-1β treatment of murine chondrocytes that were pre-incubated with 100ng/ml rhBMP5 and 10 μM PD98059 (inhibitor of ERK) showed reduced levels of total and phosphorylated p38 and ERK1/2 proteins, cleaved caspase-3, and p16 proteins compared to their corresponding controls (Figure 6C). These results demonstrate that BMP5 promotes chondrocyte senescence and apoptosis via p38/ERK signaling pathway.

Human knee cartilage samples

We obtained healthy human knee cartilage samples from patients undergoing amputation due to trauma (n=6) and OA cartilage specimens (n=6) from OA patients undergoing total knee joint arthroplasty at our hospital. The joint fluid was harvest from human osteoarthritic (n=5) and healthy (n = 5). This study was approved by the Ethics Committee of the third Affiliated Hospital of Southern Medical University. We also obtained informed consent from all patients before obtaining the cartilage samples.

Cell lines, primary chondrocyte cultures, and siRNA interference

We isolated primary chondrocytes from the rib cartilage of newborn mice as previously described [49]. The primary chondrocytes were cultured in maintenance medium consisting of DMEM and nutrient mixture F12 (DMEM:F12; Gibco, Gaithersburg, MD, USA) supplemented with 20% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) in a humidified incubator maintained at 5% CO₂ and 37° C. Primary chondrocytes were transfected with siRNA against BMP5 (BMP5-siRNA 1, 2 or 3) and control siRNA (si-NC) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The medium was changed at 6 h after transfection. The BMP5 siRNA sequences were as follows:

siRNA1 forward primer: 5′- CCAGCCUACAUGAUACCAATT-3′;

siRNA1 reverse primer: 5′- UUGGUAUCAUGUAGGCUGGTT-3′;

siRNA2 forward primer: 5′-GCUGGGUUCAAGUGGGUUATT-3′;

siRNA2 reverse primer: 5′- UAACCCACUUGAACCCAGCTT-3′;

siRNA3 forward primer: 5′- CCAGGGAAACAAGCAUCUUTT-3′;

siRNA3 reverse primer: 5′- AAGAUGCUUGUUUCCCUGGTT-3′;

Establishment of the DMM-OA model mice

We purchased thirty 12-week-old male C57BL/6J mice from the Experimental Animal Centre of Southern Medical University (Guangzhou, China). All mice were anesthetized by injecting chloral hydrate intraperitoneally followed by surgical destabilization of the medial meniscus (DMM) on the right knees to induce knee joint instability and posttraumatic osteoarthritis (PTOA) as previously described [50]. Sham surgery was performed by cutting the skin without exposing the knee joint capsule on the right knees of the control mice. DMM surgery was confirmed by using a stereo microscope to determine complete transection of the medial meniscotibial ligament (MMTL) and destabilization of the medial meniscus. The mice were randomly separated into three groups. The group 1 (n=5) animals included those that underwent sham operation and received intra-articular injection of phosphate-buffered saline; group 2 (n=5) animals included those that underwent DMM surgery and received intra-articular injection (6 μL) of 10⁸ TU/mL lentivirus-siNC (5’-3’ TTCTCCGAACGTGTCACGT); group 3 (n=5) animals included those that underwent DMM surgery and received intra-articular injection (6 μL) of 10⁸ TU/mL of lentivirus-siBMP5 (5’-3’ CCAGCCTACATGATACCAA). The lentiviruses carrying si-NC and si-BMP5 were obtained from GenePharma (Shanghai, China).

Histological analysis

The mice were sacrificed at 2 or 4 weeks after DMM surgery. Knee joints were harvested, decalcified with 0.5 M EDTA (pH 7.4) for 4 weeks, and embedded in paraffin. Then, 4 μm thick knee joint tissue sections were cut in a sagittal orientation and subjected to hematoxylin and eosin (H&E), Toluidine Blue Staining Kit (Leagene, Beijing, China) and Safranin O-Fast Green staining. Briefly, sections were deparaffinized, rehydrated, and incubated with Weigert’s iron hematoxylin solution for 3 min. Then, the stained slides were rinsed in distilled water and incubated with 1% acid–70%alcohol for 15 s. The slides were then incubated with 0.02% aqueous Fast Green solution for 5 min followed by 1% acetic acid for 30 s. Then, the slides were rinsed with distilled water and incubated with 1% Safranin-O solution for 10 min. Subsequently, the slides were dehydrated, cleared, and mounted. The severity of cartilage degeneration was assessed using the OARSI osteoarthritis cartilage histopathology assessment system [51] as shown in Table 1.

Immunohistochemistry

The de-paraffinized knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against BMP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Runx2, Col2a, MMP13, Adamts5 (Abcam, Cambridge, UK), as well as p16 and p21 (Proteintech, Rosemont, IL, USA). The slides were then incubated with the secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore Pike, USA, 1:200)) in blocking solution for 1 h at room temperature, and developed with 3,3′-diamino-benzidine (DAB, ZSGB-Bio, Beijing, China).

Immunofluorescence staining

The knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against γH2AX (Cell Signaling Technology, Danvers, MA, 1:100) and Col2a1(Abcam, Cambridge, UK). Then, the sections were incubated with species-matched Alexa-488 or Alexa-594-labeled secondary antibodies (Life Technologies, Grand Island, NY, USA). Subsequently, the sections were mounted with PBS containing DAPI (Thermo Fisher Scientific), and photographed using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). The proportions of positively stained cells relative to the total number of cells were determined for each sample.

Quantitative real-time PCR analysis

Total RNA was isolated from 90% confluent cells grown in 12-well plates using the TRIzol reagent (Life Technologies, Grand Island, NY. Equal amounts of RNA samples were reverse transcribed to generate cDNA using the Reverse transcription kit (Vazyme Biotech, Nanjing, China). The quantitative PCR (qPCR) assays were performed to determine the levels of Bmp5, TNF-α, IL-1β, SOX9, COL2A, MMP13, and ADAMTS mRNAs relative to GAPDH mRNA (internal loading control) using the Real-Time PCR Mix (Vazyme Biotech) in a Light Cycler (Roche Molecular Biochemicals, Indianapolis, IN, USA). The following primers were used for q-PCR:

BMP5 forward primer: 5′- CCACGTACCAAAACCTTGCT-3′;

BMP5 reverse primer: 5′- CAATGCTGTTAAGGCGAACA-3′;

TNF-α forward primer: 5′-ATGAGCACAGAAAGCATGA-3′;

TNF-α reverse primer: 5′-AGTAGACAGAAGAGCGTGGT-3′;

IL-1β forward primer: 5′- GCACTACAGGCTCCGAGATGAAC-3′;

IL-1β reverse primer: 5′- TTGTCGTTGCTTGGTTCTCCTTGT-3′;

SOX9 forward primer: 5′-AGTACCCGCATCTGCACAAC-3′;

SOX9 reverse primer: 5′-ACGAAGGGTCTCTTCTCGCT-3′;

ACAN forward primer: 5′- GGCAACCTCCTGGGTGTAAG-3′;

ACAN reverse primer: 5′- TGGGGTTCGTGGGCTCACAA-3′;

Col2a1 forward primer: 5′-CACACTGGTAAGTGGGGCAAGACCG-3′;

Col2a1 reverse primer: 5′-GGATTGTGTTGTTTCAGGGTTCGGG-3′;

MMP-13 forward primer: 5′-GCTGCGGTTCACTTTGAGAA-3′;

MMP-13 reverse primer, 5′-GGCGGGGATAATCTTTGTCCA-3′;

GAPDH forward primer: 5′- AAATGGTGAAGGTCGGTGTGAAC-3′;

GAPDH reverse primer, 5′- CAACAATCTCCACTTTGCCACTG-3′.

Western blotting

Total protein was extracted from chondrocytes by incubating them for 5 min at 95° C in lysis buffer [62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 50 Mm DTT, and 0.01% bromophenol blue]. Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto a nitrocellulose (NC) membrane (Bio-Rad, Hercules, CA, USA). The blots were incubated overnight at 4° C with primary antibodies against BMP5 (ABclonal, Woburn, MA, USA), RUNX2 (ABclonal), MMP13 (Proteintech), p16 (Proteintech), p53 (Proteintech), p21 (Proteintech), ERK (Cell Signaling Technology, Danvers, MA, USA), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology), and p-p38 (Cell Signaling Technology). Then, the blots were incubated at room temperature for 30 min with the corresponding HRP-labeled secondary antibodies ((Jackson ImmunoResearch Laboratories, Baltimore Pike, USA). The blots were then developed with the Enhanced chemiluminescence kit (Millipore, Burlington, MA, USA) and visualized using the Image J software.

Senescence-associated β-galactosidase (SA-β-Gal) assay

The chondrocytes were stained with the SA-β-Gal staining kit (Biovision, Milpitas, CA, USA) according to the manufacturer's protocol. SA-β-Gal-positive cells were counted in four randomly selected fields per treatment (n=5) in each sample.

TUNEL staining

DNA damage was detected by TUNEL staining in primary chondrocytes or knee cartilage sections using the In situ cell death detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The fixed cells or sections were stained for 30 min at 37° C and the nuclei were stained with DAPI. The slides were observed under an Olympus fluorescence microscope (Olympus). We randomly selected twenty-five fields in each slide and counted the numbers of TUNEL-positive cells relative to total number of cells in a field.

Apoptotic flow cytometry assay

Chondrocytes were seeded into 6-well plates at a density of 1 × 10⁵ cells per well. Flow cytometry was performed to detect the apoptosis rates of chondrocytes using the Annexin V/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Chondrocytes were washed twice cold PBS, and then resuspended in binding buffer and incubated with 5μl FITC-Annexin V and 5μl PI at room temperature for 15 min in the dark. Next, cell suspensions were determined by using the FACScan flow cytometry system (Becton Dickinson, San Diego, CA, USA).

Statistical analysis

Statistical analysis was performed using the SPSS statistical software for Windows, version 20.0 (SPSS, Chicago, IL, USA). All data were expressed as means ± SD. All experiments were performed in triplicate. The differences between groups were analyzed using one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.