Bi-directional regulation of cartilage metabolism by inhibiting BET proteins—analysis of the effect of I-BET151 on human chondrocytes and murine joints

Male 129 S1/SvImJ mice (8 weeks) were obtained from the Model Animal Research Center of Nanjing University. All animal experimental procedures were conducted in compliance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Nanjing University. Animals were maintained on standard rodent chow and had free access to food and water. All reagents were purchased from Sigma unless otherwise indicated. Dimethyl sulfoxide (DMSO) was used as the vehicle for I-BET151, IL-1β, and TNF-α.

The surgical procedures for destabilization of the medial meniscus (DMM) surgical models in the mouse have been described previously [20]. In this study, each group had 10 animals. The operations were performed under a surgical microscope. DMM surgical models were created through transecting medial meniscotibial ligament. Sham operations were identical except that the ligament was not transected. Sham operations were performed on independent mouse. All the operations were performed when the animals were at age of 10 weeks. To assess therapeutic effect of I-BET151, either I-BET151 (10 mg/kg) or DMSO was administered by intraperitoneal injection (once per day) for test or control group from 2 days after the operation to 1 day before sacrifice. At 6 and 8 weeks after treatment, the mice were sacrificed. All the mice survived until being sacrificed. The knee joints were fixed in 4% paraformaldehyde for 24 h and decalcified in EDTA for 1 week. The samples then were embedded in paraffin and successive sections 5 μm thick were prepared. Sections were stained with safranin O/fast green. Histological assessment of OA was quantified by OARSI histopathology grading [21]. All the sections of each studied joint were examined and assessed independently by two blinded investigators (JD and SZ), and the score for the most severe section of each joint was recorded. A third investigator (QG) exchanged the most severe sections selected by the two blinded investigators to each other if different sections were selected for one joint, and the deriving information of each section was blind to the two investigators. The highest score for each joint from each investigator was recorded by the third investigator, and the average of two highest scores for each joint was used for analysis.

Human samples were obtained with informed consent from the donors. The study was approved by the ethical committee of Drum Tower Hospital, Medical School, Nanjing University. Fresh cartilage samples (from three patients undergoing knee replacement surgery at Drum Tower Hospital, Medical School, Nanjing University) were chopped from the lateral condyle of the operated knee. All the three patients were males and suffered with OA. The age of the patients ranges from 64 to 68 years, and none of them has a history of immunological diseases. Primary human articular chondrocytes were cultured as previously described [22]. The cells were passaged at a 1:2 dilution, and the second to the fourth passages were used for assays.

For the cellular assays, the cells were grown to approximately 80% confluence and then starved (in medium containing 0.1% FBS) for 12 h. I-BET151 (1 μM; TOCRIS, R&D Systems) or DMSO was added 1 h prior to treatment with IL-1β (10 ng/ml; R&D Systems) or TNF-α (10 ng/ml; R&D Systems). The dose of I-BET151 was applied in the recent report about the effect of I-BET151 on rheumatoid arthritis synovial fibroblasts, and in the same report, the authors found that delayed treatment with I-BET151 showed lower effect than that pretreatment with I-BET151 [14]. The dose of IL-1β and TNF-α was mentioned in the previous studies of chondrocytes [23, 24].

The total RNA from primary human chondrocytes was isolated using TRIzol reagent (Ambion, Invitrogen) after 24 h of treatment. First-strand cDNA was prepared by reverse transcription using the PrimeScript RT Reagent Kit according to the manufacturer’s manual (TaKaRa). Real-time PCR was performed in an ABI StepOnePlus instrument (Applied Biosystems) using SYBR Green PCR Master Mix (Thermo Scientific). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of each sample was set as 1, and the relative expression of other genes was calculated and recorded. The primers used in this study are listed in Table 1.

Western blot assays were carried out as previously described [25]. The protein from primary human chondrocytes was isolated after 24 h of treatment. The primary antibodies used were rabbit polyclonal anti-MMP1 (1:500), anti-MMP2 (1:500), anti-MMP13 (1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000), goat polyclonal anti-MMP3 (1:500), mouse monoclonal anti-MMP9 (1:1000; Santa Cruz Biotechnology, Inc.), and anti-SRY-related high mobility group-box gene9 (SOX9) (1:2000; Millipore).

All data were expressed as the means S.E. and represent at least three independent experiments. Statistical comparisons were made using Student’s t test or one-way ANOVA with post hoc Tukey test. P < 0.05 was considered statistically significant. As the increase of MMP1, MMP3, MMP13, and ADAMTS4 mRNA expression by stimulation of IL-1β and TNF-α was high and variable, we used log₂ of relative expression of these four genes for data presentation and statistical analysis.

In order to check the effect of I-BET151 on OA in vivo, we examined whether I-BET151 could exert an effect on protecting articular cartilage from degeneration during OA development in a surgical mouse model of OA. I-BET151 or vehicle was administered by intraperitoneal injection for DMM and sham group, respectively. After surgery for 6 weeks, an apparent damage of articular cartilage was observed in DMM groups comparing with sham groups. As expected, I-BET151 significantly alleviated the extent of damage of articular cartilage in the DMM groups (Fig. 1a, b). After surgery for 8 weeks, an apparent damage of articular cartilage was observed in DMM groups, but in both the DMM groups and the sham groups, the extent of damage of articular cartilage showed no significant difference between I-BET151-treated group and control group (Fig. 1c, d).

Since it was believed that IL-1β- and TNF-α-induced upregulation of matrix-degrading enzymes in articular chondrocytes played critical roles in OA development [1‐3], we therefore investigated the roles of BET family proteins in the transcriptional activation of IL-1β- and TNF-α-induced matrix-degrading enzyme genes in chondrocytes. To this aim, human OA chondrocytes were pretreated with I-BET151, before exposure to IL-1β and TNF-α. Our results from a real-time PCR assay showed that MMP1, MMP3, MMP13, and ADAMTS4 mRNA expression was robustly induced by IL-1β or TNF-α, and this induction was profoundly inhibited by I-BET151 (Fig. 2a–d). In response to IL-1β or TNF-α stimulation, a weak upregulation of MMP2, MMP9, and ADAMTS5 gene expression was observed. I-BET151 repressed inducible transcription of MMP2, MMP9, and ADAMTS5 (Fig. 2e–g). However, the repression of MMP9 expression induced by TNF-α showed no statistical significance (Fig. 2f). Only ADAMTS4 basal expression was significantly repressed by I-BET151 (Fig. 2a–g).

The results from Western blot showed that IL-1β and TNF-α strongly upregulated MMP1, MMP3, and MMP13 protein expression in human chondrocytes, and this upregulation was significantly reduced by I-BET151 (Fig. 3a). The protein expression of MMP2 and MMP9 was not significantly altered by IL-1β, TNF-α, and I-BET151, respectively (Fig. 3a).

We next examined the effect of I-BET151 on regulating matrix-degrading enzyme activity. We performed gelatin zymography assays using culture supernatants. Based on MMP molecular weight, the bands represented gelatinolytic activity of specific MMP. A strong induction of low molecular weight activity might indicate MMP1, MMP3, and MMP13, which was significantly reduced when the cells were pretreated with I-BET151. By contrast, the gelatinolytic activity of MMP2 and MMP9 was not significantly altered by IL-1β, TNF-α, and I-BET151, respectively (Fig. 3b). We next tested ADAMTS activity. To this end, culture supernatants were collected for aggrecanase activity analysis. I-BET151 abolished the IL-1β- or TNF-α-induced increase in the supernatant levels of enzyme activity (Fig. 3c).

We investigated the roles of BET family proteins in the transcriptional activation of two cartilage matrix genes, aggrecan (ACAN) and collagen, type II, alpha 1 (COL2A1). The same RNA used above was applied for real-time PCR assay. The results showed that ACAN and COL2A1 expression was inhibited by IL-1β or TNF-α, and this inhibition was significant aggravated by I-BET151 (Fig. 4a, b). We also investigated the expression of SRY-box 9 (SOX9) as SOX9 was an essential transcriptional factor for chondrocyte-specific genes [26]. And we found that I-BET151 could inhibit the expression of SOX9 in human OA chondrocytes (Fig. 4c). The results of Western blot also showed that SOX9 was significantly inhibited by I-BET151 (Fig. 4d).