BRD4 has dual effects on the HMGB1 and NF-κB signalling pathways and is a potential therapeutic target for osteoarthritis

BBA - Molecular Basis of Disease 1863 (2017) 3001–3015

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BRD4 has dual effects on the HMGB1 and NF-κB signalling pathways and is a potential therapeutic target for osteoarthritis

MARK

Jiang Yafeia,b,1, Zhu Liboa,1, Zhang Taoa,b, Lu Haiminga, Wang Conga, Xue Baoa, Xu Xuna, Liu Yua, Cai Zhengdonga,b, Sang Weilina,⁎, Hua Yingqia,b,⁎, Ma Jinzhonga,⁎ a b

Department of Orthopaedics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China Shanghai Bone Tumour Institution, Shanghai 201620, China

A R T I C L E I N F O

A B S T R A C T

Keywords: BRD4 HMGB1 Inflammation Osteoarthritis Cartilage

Osteoarthritis (OA) has traditionally been defined as a non-inflammatory disease. Recently, many studies have demonstrated that OA also has an inflammatory component. BRD4, a member of the Bromodomain and ExtraTerminal Domain family, has emerged as an important regulator of some chronic inflammatory diseases. JQ1, an antagonist of BRD4, modulates transcription of several genes. Our study demonstrated that BRD4 is up-regulated in articular cartilage of OA. BRD4 inhibition attenuated the inflammation and catabolism of chondrocytes and suppressed NF-κB signalling pathway activation. In addition, BRD4 inhibition abolished the transcriptional activity of High Mobility Group Protein B1 (HMGB1). We identified HMGB1 as a direct target of BRD4. Genetic and pharmacological inhibition of BRD4 suppressed IL-1β-induced expression and translocation of HMGB1. Chromatin immunoprecipitation (ChIP) showed the enrichment of BRD4 around the HMGB1 upstream nonpromoter region, which diminished with JQ1 treatment. Finally, haematoxylin & eosin and Safranin o/Fast Green staining demonstrated that JQ1 attenuates cartilage destruction in mice with anterior cruciate ligament transection without significant toxic effects. These studies highlighted the importance of BRD4 in the chronic inflammatory reactions of OA, which, as far as we know, was the first report of this finding, and suggested that BRD4 might be a novel potential therapeutic target for the treatment of OA.

1. Introduction Osteoarthritis (OA) is a very common age-related degenerative disease, causing severe joint pain, swelling and functional impairment, and is currently considered one of the most significant causes of disability among aging populations worldwide [1]. The prevalence of OA increases with age, and a substantial proportion of adults experience pain related to OA during their lifetime [2]. Increasing evidence has demonstrated that OA, as an tissue-level failure [3], involves not only the articular cartilage surface but also adjacent structures, including the subchondral bone, ligament, capsule, synovial membrane, and periarticular muscles [4]. The hyaline articular cartilage is eroded and degraded, and subsequent remodeling of the subchondral bone with osteophyte formation around the joint functionally impedes the entire joint [5]. Despite its high morbidity rates and global burden, the disease aetiology remains poorly understood [6,7]. Thus, elucidating the aetiology and biology of OA would help identify novel potential

therapeutic targets. Epigenetic modification refers to changes in cell phenotypes that occur independently of modifications to the DNA sequence and connects chromatin structure with gene expression changes [8]. The Bromo and Extra-Terminal (BET) domain family, belongs to the family of epigenetic reader proteins [9], is characterised by the presence of two tandem bromodomains and an extra-terminal domain, which involves in diverse transcriptional networks as effectors of signal transduction [10]. The mammalian BET family comprises four paralogous genes BRD2 (originally named RING3), BRD3 (ORFX), BRD4 (MCAP) and BRDT [11]. BET proteins play a crucial role in regulating gene transcription through epigenetic interactions between bromodomains and acetylated histones during cellular proliferation and differentiation processes [10]. BRD4 is a ubiquitously expressed protein of the BET family that plays a critical role in regulating transcriptional elongation of various types of genes by recognizing N-acetylation of lysine residues on histone tails [12]. Many histone marks are detected in BRD4-

Abbreviations: CCK8, cell counting kit 8; BRD4, Brodomain 4; HMGB1, high mobility group box protein 1; OA, osteoarthritis; RAGE, receptor for advanced glycation end products; MMP, matrix metalloproteinases; DAMP, damage associated molecular patterns; IL-1β, Interleukin-1β; ChIP, Chromatin immunoprecipitation ⁎ Corresponding authors. E-mail addresses: [email protected] (W. Sang), [email protected] (Y. Hua), [email protected] (J. Ma). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbadis.2017.08.009 Received 21 April 2017; Received in revised form 28 July 2017; Accepted 16 August 2017 Available online 24 August 2017 0925-4439/ © 2017 Elsevier B.V. All rights reserved.

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Our research identified promising targets for OA intervention at the transcriptional level.

associated chromatin, suggesting that BRD4 associates with diverse factors that modulate chromatin dynamics and transcription [13]. Furthermore, BRD4 participates in direct regulatory interactions with a series of DNA-binding transcription factors to affect their disease-related functions and recruits transcriptional regulatory complexes to chromatin, predominantly through recruitment of the positive transcription elongation factor P-TEFb [14–16]. Recent studies revealed that BRD4 is associated with active promoters and enhancers, and loading of BRD4 onto “super-enhancers” drives oncogenic transcription programs [17]. Thus, BRD4 is a promising therapeutic targets in multiple important malignancies, including acute myeloid leukaemia [18] and lung [19], prostate [20,21], breast [22], pancreatic [23], and colorectal cancers [24]. Increasing studies have reported the significant role of BRD4 in the pathology of inflammatory diseases. For example, BRD4 not only regulates the expression of many NF-κB–dependent inflammatory genes but also participates in the expression of inflammatory gene enhancer RNA (eRNA) synthesis [25]. JQ1, a small molecule inhibitor of BRD4, suppressed the LPS-induced expression of pro-inflammatory cytokines and attenuated inflammatory reactions in bone marrow–derived macrophages [26,27]. In bone and joint diseases, studies have demonstrated that JQ1 plays an important role in modulating the arthritic inflammation by blocking IKK-dependent NF-κB signalling pathway activation in rheumatoid arthritis, which indicates that targeting epigenetic regulators such as BRD4 might be a potential therapeutic strategy for inflammatory arthritis [28]. A recent study of anot her BET inhibitor, I-BET151, demonstrated that BET inhibition suppresses the expression of inflammatory genes and matrix-degrading enzymes in rheumatoid arthritis synovial fibroblasts [29,30]. However, the role of BRD4 in primary OA has not been studied, and thus, exploring new regulatory pathways for BRD4 would increase our knowledge of its potential role in inflammatory reactions of OA. Several genes have been identified as drivers of OA development [31,32]. High Mobility Group Box Protein 1 (HMGB1) is an ubiquitous chromatin component widely expressed in immune and other cells [33]. Acting as a pro-inflammatory cytokine itself, HMGB1 activates the NF-κB signalling pathway, which is involved in the inflammatory reactions of several diseases [34]. Studies have demonstrated that HMGB1 and its receptor, receptor for advanced glycation end products (RAGE), are both significantly increased in synovial and peripheral blood of knee osteoarthritis (KOA) patients [35–37]. Additionally, the HMGB1 levels in synovial fluid were associated with the severity of synovitis, pain, and daily activities in KOA patients [38]. HMGB1 was also related to the histopathological grade of cartilage destruction and NF-κB signalling pathway activity [39,40]. Furthermore, HMGB1 is involved in the pathogenesis of rheumatoid arthritis (RA) by binding to LPS, yielding a complex that recognizes TLRs/RAGE on synovial fibroblasts and initiates an inflammatory cascade that leads to the secretion of inflammatory cytokines and chemokines, production of tissue-destructive enzymes, and finally RA [41,42]. Despite the relationship between HMGB1 and OA, the transcriptional regulatory mechanism that results in proinflammatory reactions of HMGB1 in OA is still largely unknown. In this study, we explored the transcriptional regulation of HMGB1 in OA. NF-κB signalling is highly activated in OA [43,44], and BRD4 inhibition has been shown to be an inhibitor of inflammation in bone diseases [45,46]. HMGB1 is also a well-conserved and pivotal regulator of the immune responses and inflammation of OA. The exact mechanisms among BRD4, HMGB1 and NF-kB signalling pathway activation in OA have not been fully elucidated. Collectively, in our study, we evaluated the expression levels of BRD4 in OA cartilage and assessed the underlying mechanisms of BRD4 inhibition in human chondrocyte cell lines in vitro and in a mouse anterior cruciate ligament transection (ACLT) OA model in vivo. We showed that BRD4 could positively regulate HMGB1 and ultimately affect NF-κB signalling to influence the inflammatory reactions and catabolic events in articular chondrocytes.

2. Materials and methods 2.1. Reagents and antibodies The BRD4 bromodomain-specific inhibitor JQ1(+) was purchased from Selleck Chemicals (Houston, TX, USA) anterior cruciate ligament transection and was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and stored in the dark at −20 °C. IL-1β and all ELISA kits was obtained from R & D Systems (R & D Systems, USA). Cell culture reagents, including DMEM/high-glucose, antibiotics, and trypsin–EDTA, were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Foetal bovine serum (FBS) was obtained from Sigma Aldrich (St. Louis, MO, USA). PBS and other reagents for cell culture were purchased from Thermo (Waltham, MA, USA). Antibodies against p-p65, p65, IκBɑ, and HMGB1 were obtained from Cell Signalling (Danvers, MA, USA). BRD4, Histones H3 and GAPDH antibodies were purchased from Abcam (Hong Kong, China). β-Actin antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). 2.2. Cell line and patient tissue specimens The human chondrocyte cell line sw1353 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM/high-glucose supplemented with 10% foetal bovine serum and antibiotics at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Six pairs of fresh articular cartilages were selected from patients who underwent total knee arthroplasty for primary OA and upon total hip replacement for femur neck fracture (FNF). Another 20 KOA patients with different degrees of joint destruction according to the KellgrenLawrence scale system were employed in our studies as shown in Fig. 2. All patients underwent surgery at the Department of Orthopaedics of the Shanghai General Hospital of Shanghai JiaoTong University School of Medicine (Shanghai, China) between 2015 and 2016. The demographics of the patients in Fig. 1 are shown in Supplementary Table S1.A. The demographics of the patients in Fig. 2 are shown in Supplementary Table S1.B. The specimens were dissected from the tibial plateau, as is indicated in Fig. 2A, using a hollow drill, after removing soft tissues, the specimens were immediately frozen in liquid nitrogen and stored at − 80 °C for protein and mRNA analysis or stored in 4% PFA for micro CT and histological analysis. The present research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital, and informed consent was obtained from all patients.

2.3. Micro CT assessment Subchondral bone explants from the clinical specimens were fixed in 4% PFA for 24 h. These were then analysed using high-resolution μCT by the YUEBO Company (Hangzhou, China). Isotropic resolution was 6 μm. Then, three-dimensional (3D) reconstructions of subchondral bone were performed. The 3D images showed the whole subchondral trabecula bone of clinical bone explants, and the subchondral bone samples were used to perform the 3D histomorphometric analysis. The 3D structural parameters analysis included bone mineral density (BMD), bone volume/total tissue volume (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp) and structure model index (SMI). 2.4. Human primary chondrocytes isolation Human OA chondrocytes were isolated from OA patients who underwent total knee arthroplasty. The articular cartilage for primary chondrocytes isolation were collected from the relative normal regions 3002

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Fig. 1. BRD4 is increased in OA cartilage. (A) Relative levels of BRD4 mRNA in OA cartilage (n = 6) and normal cartilage (n = 6) were evaluated by real-time PCR; β-actin was used as an endogenous control. (B) The protein level of BRD4 in clinical cartilage samples was detected by western blot; GAPDH was used as loading control. (C) (a–b) Clinical patient cartilage samples were stained with Safranin O/Fast Green to analyse the histologic changes. (c–f) The expression of collagen II and BRD4 was detected by immunohistochemical staining. The expression of BRD4 in cartilage is shown (Right panel). (D) (a–b) Histological examination of ACLT OA mouse knee joints using Safranin O/Fast Green staining revealed rapid progression of cartilage damage in ACLT OA mice compared with the sham group (without ACLT surgery). (c–f) The expression levels of BRD4 and collagen II were measured by immunohistochemical staining. The expression of BRD4 in cartilage is shown (Right panel). Data are shown as the mean ± S.D. (*) P < 0.05 compared to relative normal cartilage.

2.5. Cell viability assay

of OA cartilage. In brief, the cartilage were washed three times using PBS, and cut into 0.1 ∗ 0.1 ∗ 0.1 cm3. After digesting with trypsin (2.5% for 30 min) and collagenase II (2 mg/ml for overnight), the primary chondrocytes were filtered by using nylon meshes (70 μm). The isolated chondrocytes were washed three times with PBS and finally incubated in DMEM/F12 culture medium containing 10% FBS. The first-passage chondrocytes were used in our experiments.

The effect of JQ1 on cell viability was determined with CCK8 assays (Dojindo, Kumamoto, Japan). Cell suspensions (5 × 103/ml) were seeded into 96-well plates overnight and then treated with various concentration of JQ1 (0, 100, 200, 400, 600, 800, 1000 nM). JQ1 was dissolved in DMSO, and DMSO concentration was maintained at < 0.05% in all wells. After 24, 48, 72 and 96 h of JQ1 exposure, 3003

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Fig. 2. The correlation of BRD4 expression levels and the severity of OA. (A) (a–d) Clinical samples of different stages of KOA were graded by the Kellgren-Lawrence scale system based on the X-ray analysis. (e–h) The micro structure of subchondral bone under the tibial plateau was analysed by micro CT. (i–l) The cartilage destruction of clinical samples was analysed by Safranin O/Fast Green staining and was classified according to the OARSI grade system. (m–p) The expression levels of collagen type II in articular cartilage were analysed by immunohistochemical staining. (q–t) The expression levels of BRD4 in articular cartilage were analysed by immunohistochemical staining. (B) The statistics of bone micro structure parameters, OARSI scores, and immunohistochemistry. Data are shown as the mean ± S.D. of three experimental replicates. (*) P < 0.05 compared with control.

10 μl CCK8 solution was added to each well, and the samples were incubated at 37 °C for 2 h. The absorption was acquired at 450 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA) to determine the cell viability. Three independent experiments were carried out in triplicate.

microscope (Olympus, Japan). The obtained images were quantitative analysed using ImageJ software (NIH, USA). 2.10. Immnohistochemical analysis The section were dewaxed with xylene and rehydrated in a graded series of ethanol. Sections were blocked with 3% H2O2 to reduce endogenous peroxide, and then, slides were prepared for antigen retrieval using enzymatic digestion with proteinase K (10 mM, Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 15 min. After blocking with 1% bovine serum albumin (BSA) for 15 min to inhibit nonspecific staining, the sections were incubated with primary antibodies against BRD4 (dilution 1:400), HMGB1 (dilution 1:400) and P65 (dilution 1:400) at 4 °C overnight, followed by incubation with a biotinylated secondary antibody. The reaction was developed using a DAB Kit (BD Bioscience, Franklin Lakes, NJ, USA), and the tissues were counterstained with haematoxylin. Blinded evaluation of immunoreactivity was executed independently by two pathologists. The proportion of immunopositive cells was evaluated.

2.6. BRD4 small interfering RNAs (siRNAs), plasmids and cell transfection Small interfering RNA (siRNA) oligonucleotides against BRD4 and the scrambled sequences were synthesized by RiboBio Company (Guangzhou, China). The following siRNA sequences are listed in Supplementary Table S1.C. Coding sequence for human BRD4 was cloned into the expression vector pCDNA3.1 (Life Technologies). Transfection was performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's recommendations. After transfection, cells were collected for RNA and protein extraction for further investigation. 2.7. RNA isolation and real-time PCR After treatment, total RNA of articular cartilage samples and sw1353 cells was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. For each sample, 1 mg of RNA was reverse-transcribed (RT) using MLV reverse transcriptase according to the instruction manual. The specific transcripts were quantified by quantitative real-time PCR using the SYBR® Premix DimerEraser™ (Perfect Real Time) (TaKaRa, Japan) and analysed by RT-qPCR in ViiA™ 7 Real-Time PCR System (Life Technology, USA) according to the manufacturer's instructions. Amplification conditions were as follows: 2 min of pre-incubation at 65 °C, 10 min at 95 °C for enzyme activation, and 40 cycles of denaturation at 95 °C for 30 s, annealing at 95 °C for 5 s, and extension at 65 °C for 30 s. The mRNA levels of target genes were normalized to the β-actin mRNA level, and the 2− ΔΔCt method was used to assess the relative expression of different candidate genes. The primers used for real time PCR are listed in Supplementary Table S1.D.

2.11. Western blot analysis After treatment, chondrocytes were washed with cold PBS three times and then lysed in ice-cold RIPA (Beyotime Biotechnology, Jiangsu, China) containing 10 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice. Total cellular protein concentration was determined using BCA assay kit (Beyotime Biotechnology, Jiangsu, China). The proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h in 5% non-fat milk in PBST buffer, the membrane was incubated with primary antibodies at 4 °C overnight, and then, the membrane was washed three times for 10 min with TBS-T solution and incubated for 1 h with the corresponding HRP-conjugated secondary antibodies (1:5000, Abgent). Chemiluminescent detection was performed using an ECL kit (Pierce Chemical, Rockford, IL, USA) and Bio-Rad ChemiDoc MP Imaging System. All experiments were performed in triplicate. Gray value of the bands was analysed by Image J2x software (NIH, USA).

2.8. ELISA 2.12. Nuclear and cytoplasmic extraction MMPs and inflammatory cytokines, especially MMP3, MMP9, MMP13, IL-6 and TNF-α, are believed to mediate the progression of OA. The sw1353 cells supernatants from different groups were collected and were analysed using ELISA kits (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The results are expressed as picograms per millilitre.

Following treatments, cells were collected and nuclear and cytoplasmic proteins were extracted with a nuclear extract kit (Beyotime, China) according to the manufacturer's instructions. The quality and purity of the subcellular fractionation was determined by immunoblotting using antibodies against cytoplasmic (β-actin) and nuclear (Histones H3) proteins to demonstrate standardization of this method.

2.9. Immunofluorescence and image quantification The cells cultured in glass-bottom dishes were fixed with 4% PFA for 30 min and incubated with blocking buffer (PBS with 10% goat serum and 1% BSA) for 1 h at room temperature. p65 (RelA) antibody (dilution at 1:400), BRD4 antibody (dilution at 1:400) and HMGB1 antibody (dilution at 1:400) in PBS was added and incubated overnight at 4 °C. After washing three times for 5 min with TBS-T solution, goat antirabbit IgG antibody coupled to Alexa Fluor 594 (Life Technologies, USA) at 1:200 dilution in PBS was incubated for 30 min at room temperature under dark conditions. After washing three times for 5 min with TBS-T solution, nuclear DNA was stained with DAPI (Dojindo, Japan) for 3 min. After washing three times for 5 min, the dish was covered with PBS, and the images were acquired using a fluorescence

2.13. Chromatin immunoprecipitation (ChIP) assay A ChIP assay was performed with a modified ChIP system (Invitrogen-Life Technologies Inc.) using 5 μg of BRD4 antibody or control rabbit IgG (R & D Systems). Real-time PCR was performed on fragmented DNA using specific primers for the HMGB1 loci. Primers were designed to amplify sites within each gene locus based on the H3K27 acetylation level, and a negative control region was designed outside the promoter region. Primers sequence are listed in Supplementary Table S1.E. Enrichment of the H3K27Ac histone mark across the genome was determined by a ChIP-seq assay on 7 cell lines from ENCODE web resources. 3005

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patient cartilage samples were stained with Safranin O/Fast Green to assess the histologic changes. OA cartilage displayed severe cartilage surface loss, loss of Safranin O staining and a significantly higher degree of destruction compared with the corresponding normal cartilage (Fig. 1C, a–b). The expression of collagen type II and BRD4 was detected using immunohistochemistry (Fig. 1C, c–f). The results showed that the samples with higher grades of OARSI were negative for Safranin O and collagen II staining, while the expression of BRD4 in these patients was up-regulated significantly (right panel) (P < 0.05). To further support our results, an ACLT OA model was established in C57BL/6 mice. Histological examination of ACLT mice showed moderate pathological osteoarthritic changes characterised by Safranin O loss and cartilage fibrillation and a significantly higher degree of destruction compared with the sham surgery group (Fig. 1D, a–b). Immunohistochemistry further indicated that ACLT mouse cartilage showed severe loss of collagen type II (Fig. 1D, c–d). Additionally, immunohistochemical results indicated that BRD4 predominantly accumulated in the cell nucleus of chondrocytes, and the expression of BRD4 in the cartilage of ACLT mice was strikingly higher than that in the normal cartilages of the sham group mice (Fig. 1D, e–f) (right panel) (P < 0.05). These results revealed that BRD4 may participate in the pathogenesis of OA.

2.14. Experimental mouse OA model and treatment Twenty C57BL/6 mice were housed in specific pathogen-free facilities at the laboratory animal unit of the Shanghai General Hospital. The mouse OA model was established by anterior cruciate ligament transection (ACLT). Following anesthesia with intraperitoneal chloral hydrate, the right knee joint skin was shaved and disinfected with a topical antiseptic. We used a needle in the right knee without opening the joint, and then, an anterior drawer test was carried out to ensure the anterior cruciate ligament has been completely transected. For postoperative analgesia, 0.02 mg/kg fentanyl citrate (Fentanyl; Abbott, Chicago, Illinois) was administered subcutaneously twice daily for 3 days after surgery. Mice were randomly divided into four groups. Group I (sham group, n = 5) mice did not undergo surgery or treatment. Group II (ACLT + vehicle, n = 5) mice underwent ACLT and intra-articular injection with PBS (10 μl, three times per week). Group III (ACLT + low dose JQ1, n = 5) mice underwent ACLT and intraarticular injection with low-dose JQ1 (12.5 mg/kg, 10 μl, three times per week). In Group IV (ACLT + high dose JQ1, n = 5), mice underwent ACLT surgery and intra-articular injection with JQ1 (25 mg/kg, 10 μl, three times per week). Mice were maintained on a 12 h light/ dark cycle under a constant temperature of 24 ± 2 °C and a relative humidity of 55% ± 5% and were allowed free access to food and water. The mice could move freely in the cages after surgery.

3.2. Correlation between BRD4 expression and the severity of OA 2.15. Histological assessments OA is a progressive joint disorder characterised by the uneven and gradual degeneration of articular cartilage, subchondral bone remodeling and sclerosis, thickening of calcified cartilage and thinning of trabeculae [49]. As shown in Fig. 2A, gross macroscopic observation indicated that OA medial tibial plateau specimens had rough articular cartilage surfaces and significant osteophyte formation around the tibial plateau. Furthermore, subchondral bone under the injured cartilage showed sclerosis. On the contrary, in the laterial compartment, the degree of cartilage damage was less pronounced, the articular surface was relatively smooth without evident osteophyte formation, and the severity of subchondral bone sclerosis was less serious than that of the medial compartment. X-ray analysis was the major method of OA diagnosis in our clinical evaluation [4]. We assessed the correlation between the expression level of BRD4 and the severity changes of knee OA cartilage. Knee OA is diagnosed based on the symptoms accompanied by X-ray radiographic changes. To facilitate objective and consistent assessments, radiographs are generally scored using the Kellgren-Lawrence scale system (Fig. 2B, a–d), which scores several features of OA in both the medial and lateral tibiofemoral compartments on an ordinal scale from zero to four [50]. The micro structure of subchondral bone was detected using micro CT (Fig. 2B, e–h). The structural integrity of the articular cartilage was detected using Safranin O/Fast Green staining (Fig. 2B, i–l) and evaluated by the Osteoarthritis Research Society International (OARSI) grading system. The expression levels of collagen type II (Fig. 2B, m–p) and BRD4 (Fig. 2B, q–t) were detected using immunohistochemical staining. Fig. 2C shows various clinical parameters from Fig. 2B, including BMD, BV/TV, Tb.N, OARSI scores, and the relative expression of BRD4 and collagen II. These results indicated that BRD4 levels in cartilage tissues gradually increased from grade I to grade IV. Furthermore, Pearson's correlation analysis was used to examine the correlation between BRD4 gene expression and the severity of OA based on the parameters from micro CT, OARSI scores and immunohistochemical staining of collagen type II. (*) P < 0.05 was considered statistically significant. A significant relationship was detected between BRD4 expression and the severity of cartilage degeneration in samples from OA patients (Table 1), which indicated that BRD4 expression may provide guidance for clinical judgment of the severity of OA.

All animals were sacrificed at 6 weeks after surgery. Knee joint samples were collected and fixed in 4% paraformaldehyde for 24 h. After decalcification in 10% EDTA for several days, the sample were embedded in paraffin, sectioned in 4 μm sections, and stained with haematoxylin & eosin (H & E) or Safranin O/Fast Green staining for further histological analysis as described previously [47]. Histopathological features were semi-quantitatively scored according to the Osteoarthritis Research Society International (OARSI) grade system [48], which comprises 6 histological grades and 4 histological stages. The total score (score = grade × stage) ranges from 1 point (normal articular cartilage) to 24 points (no repair). Three independent observers scored each section, and the scores for all of the sections were averaged within each specimen. All scorings were performed by three independent observers who were blinded to the treatment groups. All procedures for consideration of animal welfare were reviewed and approved by the ethical committee of the Shanghai General Hospital. 2.16. Statistical analysis Student's t-test was used to detect the difference between two groups. All data are presented as the mean ± SD from at least three independent experiments. The Pearson χ2 test was used to analyse the relationship between BRD4 expression and clinicopathological parameters of OA. ANOVA was used to detect the difference among three groups or more. All statistical analyses were performed using SPSS version 18.0 software (IBM Corporation, Chicago, USA). (*) P < 0.05 was considered to be statistically significant. 3. Results 3.1. BRD4 is increased in OA cartilage To assess the therapeutic potential of targeting RRD4 in osteoarthritis, we first evaluated the expression level of BRD4 in OA cartilage. We collected 6 pairs of primary knee OA cartilage samples and corresponding normal cartilage tissues from patients with femoral neck fractures. As shown in Fig. 1A and B, both BRD4 mRNA and protein levels were significantly up-regulated in cartilage of OA patients compared with those in normal cartilage tissues (P < 0.05). The clinical 3006

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different siRNAs targeting BRD4 expression or with a scrambled control (siCtrl). siRNA treatment significantly suppressed HMGB1 expression. Then, we determined whether overexpression of BRD4 would increase the expression level of HMGB1. To this end, the readily transfectable sw1353 cells were transfected with an empty vector or with a plasmid for BRD4 expression, and as expected, BRD4 overexpression dramatically increased the expression of HMGB1 (Fig. 4E). We postulated that BRD4 might regulate HMGB1 expression in chondrocytes in OA, possibly through direct effects on the HMGB1 promoter region. To determine whether BRD4 binds directly to the HMGB1 locus, we performed ChIP assays using an antibody against BRD4 in sw1353 cells. The increased enrichment for histone 3 lysine 27 acetylation (H3K27ac) at the HMGB1 promoter and upstream nonpromoter region indicates active transcription (Fig. 4F), perhaps associated with a “super-enhancer region” as recently described [52]. We found that BRD4 was enriched in upstream non-promoter region of HMGB1. Furthermore, treatment with JQ1 induced the release of BRD4 from the non-promoter and binding sites of HMGB1 (Fig. 4F).

Table 1 Correlation between BRD4 expression level and clinical data. Clinical samples parameters

BV/TV BMD Tb.N OARSI scores Collagen II

BRD4 expression level Pearson R value

P value

0.7922 − 0.8192 0.8579 0.8522 − 0.5794

< 0.05 < 0.05 < 0.05 < 0.05 < 0.05

3.3. BRD4 inhibition suppresses the production of pro-inflammatory cytokines and catabolic factors induced by IL-1β in chondrocyte Given that BRD4 is overexpressed in OA articular cartilages in our study, we further explored whether inhibition of BRD4 could affect normal chondrocyte activity. JQ1, as shown in Fig. 3A, is a broadspectrum inhibitor of the BET family, which can target the BD domains of BRD4 with high affinity. We first used CCK8 to test the viability of sw1353 chondrocytes pretreated with JQ1 at concentrations ranging from 50 to 1000 nM. JQ1 inhibited cell viability in a dose-dependent manner, and up to 400 nM, this compound had no significant cytotoxic effects on the chondrocytes (Fig. 3B). To investigate the mechanism underlying the effects of BRD4 on chondrocyte inflammation and catabolism, sw1353 chondrocytes were stimulated with IL-1β to induce the expression of inflammatory cytokines and catabolic genes, including IL-6, IL-8, IL-10, TNF-α, MMP2, MMP3, MMP9 and MMP13. This system mimics primary human chondrocytes and is an in vitro substitute for osteoarthritis [51]. Realtime PCR showed that IL-1β increased the expression of these genes at the transcriptional level, while pre-treatment with JQ1 reversed this process in a concentration-dependent manner (Fig. 3C). To support this, we used primary chondrocytes to further verify the results of real-time PCR, and we found that the results was basically consistent with that of sw1353 cells (Supplementary text 1A). To further validate the above results, we also assessed the effects of BRD4 inhibition on the secretion levels of MMP-3, MMP-9, MMP-13, IL6 and TNF-ɑ in the supernatant by ELISA (Fig. 3D and E). The results further supported the real-time PCR results. Collectively, these results indicated that BRD4 plays an important role in the regulation of inflammatory reactions of OA, which may provide a new direction for the treatment of OA in the future.

3.5. BRD4 regulates IL-1β-induced HMGB1 expression and translocation from the nucleus to the cytoplasm in chondrocytes To investigate the specific molecular mechanism of BRD4 regulation of HMGB1 in OA, we established a model of OA in vitro. The chondrosarcoma sw1353 cells expressed a comparable, but not identical, set of genes to those of primary chondrocytes when they were stimulated with IL-1β [53], which was why we used sw1353 cells pretreated with IL-1β as a substitute for primary human chondrocytes and an in vitro model of OA. Real-time PCR (Fig. 5A) and western blot analysis (Fig. 5B) showed that BRD4 and HMGB1 were both up-regulated following treatment with IL-1β in a dose-dependent manner. Furthermore, immunofluorescence also supported these results, showing that the expression level of BRD4 was up-regulated in the presence of IL-1β and was predominantly located in the nucleus (Fig. 5C). Fig. 5D is a statistical panel of Fig. 5C. To demonstrate the roles of BRD4 in IL-1β-induced HMGB1 expression in chondrocytes, we used JQ1 to suppress the activity of BRD4. Real-time PCR results showed that JQ1 significantly impaired the expression level of HMGB1 induced by IL-1β in a dose-dependent manner (Fig. 5E). Western blot analysis further supported these results (Fig. 5F). We knocked down BRD4 (Fig. 5G) to determine the effect of BRD4 on IL-1β-induced HMGB1 expression. We observed a sharp decline in IL1β-induced HMGB1 expression after BRD4 knockdown (Fig. 5H). Therefore, we deduced that IL-1β-induced HMGB1 production by chondrocytes is dependent on BRD4. In unstimulated conditions, HMGB1 is predominantly located in the nucleus, while after stimulation with IL-1β, HMGB1 migrates from the nucleus to the cytoplasm. Our immunofluorescence results showed that BRD4 knockdown strongly suppressed the IL-1β-induced translocation of HMGB1 from the nucleus to the cytoplasm (Fig. 5I). These results indicated that the IL-1β-induced up-regulation and translocation of HMGB1 from the nucleus to the cytoplasm was dependent on the presence of BRD4 in chondrocytes.

3.4. HMGB1 is a direct target of BRD4 in chondrocytes Previous studies have demonstrated that HMGB1 was highly expressed in OA cartilage and was associated with the severity of OA. We first detected the expression levels of HMGB1 and evaluated the potential correlation between BRD4 and HMGB1 in OA cartilages using western blotting (Fig. 4A). Semi-quantitative analysis of western blotting showed a strong correlation between the expression level of BRD4 and HMGB1 (Fig. 4B) (P < 0.05). As HMGB1 is a specific damage associated molecular pattern (DAMP) of OA [35], determining whether BRD4 influences the expression level of HMGB1 and cartilage destruction is important. In this regard, we performed real-time PCR and western blot analysis and found that BRD4 inhibition with the highly selective inhibitor JQ1 dramatically decreased the mRNA (Fig. 4C) and protein (Fig. 4D) levels of HMGB1 in a time- and dose-dependent manner. We then used primary chondrocytes to further verify the results of real-time PCR, and we found that the results was basically consistent with that of sw1353 cells (Supplementary text 1B). Furthermore, to firmly establish a role of BRD4 in the expression of HMGB1, we performed RNAi studies to determine whether genetic suppression of BRD4 would have similar effects on the expression of HMGB1. sw1353 cells were treated with two

3.6. BRD4 inhibition suppresses IL-1β-induced NF-κB signalling pathway activation In recent years, inflammation has been shown to be involved in OA pathology. The NF-κB signalling pathway, which is highly activated in OA, is a canonical pro-inflammatory signalling pathway. Numerous proinflammatory mediators, including IL-1β, IL-6 and TNF-ɑ, are detected in synovial fluid or at the disease site, where they participate in inflammatory reactions by activating the NF-κB signalling pathway and promoting the expression of numerous genes involved in the regulation of the innate and adaptive immune responses. The NF-κB signalling pathway was highly activated in OA (Fig. 6A) and has been implicated as a key regulator in cartilage destruction and 3007

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Fig. 3. BRD4 inhibition attenuates the expression of pro-inflammatory cytokines and matrix metalloproteinases in chondrocytes. (A) The molecular structure of JQ1. (B) Effects of JQ1 on cell viability of chondrocytes were measured by CCK8 assays. (C) The effects of JQ1 on MMP-2, -3, -9, -13, IL-6, -8, -10 and TNF-ɑ gene expression induced by IL-1β in sw1353 chondrocytes. Chondrocytes were pretreated with JQ1 (200, 400 nM) for 2 h, followed by stimulation with IL-1β (10 ng/ml) for 24 h. Levels of gene expression were determined by realtime PCR. (D) The protein levels of MMP3,MMP9 and MMP13 in the supernatant were assessed by ELISAs. (E) The expression levels of IL-6 and TNF-ɑ in conditioned medium were assessed by ELISAs. (*) P < 0.05 compared with control.

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Fig. 4. HMGB1 is a direct target of BRD4 in chondrocytes. (A) The expression of BRD4 and HMGB1 in each patient was measured using western blotting. GAPDH was used as loading control. (B) Correlation between BRD4 and HMGB1 expression in human OA cartilage specimens was tested by Pearson's correlation test. P < 0.05 was considered statistically significant. sw1353 chondrocytes were pre-treated with different concentrations of JQ1 for 24 h or 200 nM JQ1 for different time points, and the expression of HMGB1 was analysed by (C) real-time PCR and (D) western blot. (E) sw1353 cells were transfected with BRD4 siRNA or BRD4 plasmid, and the expression level of HMGB1 was analysed by western blot. (F) ChIP with a BRD4 antibody at the HMGB1 upstream non-promoter region in cells treated with 500 nM JQ1 for 4 h. Enrichment is shown as the percentage of total input DNA. The top track shows the levels of enrichment of the H3K27Ac histone mark across the genome as determined by a ChIP assay of 7 cell lines from ENCODE.

was reversed by both BRD4 siRNA and JQ1 pre-treatment (Fig. 6B and C). We then used human primary chondrocytes to further verify the results of Fig. 6B (Supplementary text 1C). Our nuclear cytoplasmic separation and immunofluorescence results further support these findings, while JQ1 abolished the IL-1β-induced translocation of p65 from the cytoplasm to the nucleus (Fig. 6D and E). These observations demonstrated that BRD4 may play an important role in the activation of the NF-κB signalling pathway, activate the inflammatory cascade at the whole cell level, and accelerate the progression of osteoarthritis. Thus, inhibition of BRD4 is expected to reverse this process and provides a new direction for clinical prevention and treatment.

bone remodeling of OA. Once activated by the extracellular signals, the phosphorylation and degradation of IκBα promotes NF-κB P65 translocation from the cytoplasm to the nucleus and activates target gene transcription. In our experiment, we examined the effects of BRD4 inhibition on the activation of the NF-κB signalling pathway in a cellular model of OA. First, we used western blot analysis to determine whether JQ1 or BRD4 siRNA inhibits IL-1β-induced NF-κB signalling pathway activation. Our results showed that nuclear NF-κB p65 was markedly increased after exposure to IL-1β alone and was reduced by BRD4 siRNA or JQ1 pre-treatment in a dose-dependent manner. Additionally, IκBα was substantially degraded 30 min after IL-1β treatment, which 3009

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Fig. 5. BRD4 mediates IL-1β-induced expression and translocation of HMGB1 in chondrocytes. sw1353 chondrocytes were exposed to IL-1β (10 ng/ml) for 24 h, and the expression level of BRD4 was measured by (A) real-time PCR, (B) western blot and (C) immunofluorescence. (D) Panel showing quantitative analysis of BRD4 in the nucleus of sw1353 chondrocytes. (E, F) JQ1 decreased IL-1β-induced production of HMGB1 in chondrocytes. Chondrocytes pre-treated with different doses of JQ1 or vehicle were exposed to 10 ng/ml IL-1β for 24 h, and the expression of HMGB1 was determined by real-time PCR and western blot. (G) BRD4 in sw1353 was depleted successfully using BRD4-specific siRNA. SW1353 cells were transfected with sc siRNA or BRD4-specific siRNA. After 48 h, the cells were treated with IL-1β (10 ng/ml) for the indicated periods. (H) Depletion of BRD4 negatively regulated IL-1β-induced expression and translocation of HMGB1 in sw1353 cells. HMGB1 expression was measured with western blot; GAPDH was used as a loading control. (I) The translocation of HMGB1 in sw1353 cells was determined by indirect immunofluorescence. Scale bar = 200 μm. Data are shown as the mean ± S.D. of three independent experiments. (*) P < 0.05 compared with the control group.

high-dose JQ1 intro-articular injection groups (Fig. 7A, i–p). Based on the immunostaining intensities of five randomly selected areas of the articular cartilage, the expression levels of HMGB1 and P65 were calculated and analysed (Fig. 7B).

3.7. BRD4 inhibition inhibits the expression of HMGB1 and attenuates the activation of the NF-κB signalling pathway in vivo We examined the biological effects of BRD4 inhibition on HMGB1 gene expression and on the activity of the NF-κB signalling pathway using a mouse ACLT OA model. As shown in Fig. 7, the expression levels of HMGB1 were increased in the ACLT + PBS intro-articular injection group compared to the sham surgery group, while JQ1 decreased the expression levels of HMGB1 in a concentration-dependent manner (Fig. 7A, a–h). The results of NF-κB p65 immnohistochemical staining revealed that percentages of p65-positive chondrocytes in the ACLT with PBS groups were substantially higher than those in the sham surgery groups, while no such change was observed in the low-dose and

3.8. JQ1 rescues cartilage destruction without significant toxicity in a mouse OA model in vivo Given the observations in vitro, the effects of BRD4 inhibition on articular cartilage protection of OA were assessed in a mouse ACLT model. Histologic analysis using H & E and Safranin O/Fast Green staining showed the cartilage protective effects of JQ1 in a mouse ACLT OA model. In PBS-treated mice, total erosion of cartilage was severe 3010

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Fig. 6. BRD4 inhibition suppresses the activation of the NFκB signalling pathway induced by IL-1β. (A) The protein level of P-P65 in clinical cartilage samples was detected by western blot; GAPDH was used as loading control. SW1353 chondrocytes were pretreated with different concentrations of JQ1 (B) (200, 400 and 600 nM) for 2 h or BRD4 siRNA (C) for 48 h, then stimulated with IL-1β (10 ng/ml) for 30 min. Related proteins in the NF-κB signalling pathway, IκBα, p65 and their phosphorylated protein levels were analysed by western blot; GAPDH was used as loading control. (D) Nuclear and cytoplasmic proteins were extracted, and p65 and their phosphorylated protein levels in the nucleus and cytoplasm were analysed by western blot. (E) Immunofluorescence staining showing the p65 distribution in sw1353 cells; nuclei were stained by DAPI (blue). Quantitative analysis of P65 in the nuclear of sw1353 chondrocytes (right panel). Values are shown as the mean ± S.D. (*) P < 0.05 vs control.

mice in our experiments.

and could even be observed down to subchondral bone in some cases, while JQ1-treated animals showed less severe destruction, which predominantly appeared as loss of Safranin O staining and surface irregularity (Fig. 8A). Articular cartilage histopathology scores of Safranin O/Fast Green-stained sections from the different treatment groups are presented. We found that PBS-treated ACLT mice displayed severe cartilage surface loss and clefts and exhibited a significantly higher degree of destruction compared to sham controls, while JQ1 significantly attenuated the degradation of cartilage in a dose-dependent manner (Fig. 8B). To investigate the systemic potential toxicity of JQ1, the body weight of the mice was detected twice a week over the course of the experiment, and the major organs, including heart, liver, spleen, lung and kidney, were paraffin-embedded for H & E staining. We found no significant changes in mouse body weight after intro-articular injection with JQ1 compared with PBS-treated mice (Fig. 8C). H & E staining revealed that JQ1 resulted in no obvious damage to major organs (Fig. 8D). These findings indicated that JQ1 had few side effects on the

4. Discussion OA has traditionally been regarded as a degenerative disease. However, it is well established that OA is an organ level disease with complicated mechanisms that involve chronic systemic low grade inflammation [49,54]. Active catabolism and aseptic inflammation play important roles during the development of the disease. Stiffness and restricted motion are the most significant causes of global disability, which are also major barriers to improving the quality of life of elderly people [2]. Progressive degeneration of articular cartilage and joint space are observed, leading to the whole joint function loss and subsequent total joint arthroplasty. Current treatments are primarily focused on reducing clinical symptoms, and there are few effective therapies [55]. Further elucidating the molecular mechanism of this disease may identify new promising therapeutic targets for OA. OA is an inflammatory disorder involving degradation of articular 3011

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Fig. 7. JQ1 inhibits the expression of HMGB1 and attenuates the activation of the NF-κB signalling pathway in vivo. (A) The expression of HMGB1 and the activity of the NF-κB signalling pathway (p65 expression level) in the articular cartilage was assessed by immunohistochemical staining (n = 5); (B) The panels show the quantification of immunohistochemical staining (n = 5). Scale bar = 800 μm (top), 200 μm (bottom). (*) P < 0.05 vs model group.

in inflammation, and BRD4 inhibition treatment has been shown to have anti-rheumatoid arthritis (RA) effects in vitro and in vivo [28,61]. However, little is known about its effects on primary OA. Our data suggest for the first time that the expression level of BRD4 was positively correlated with the severity of OA. We demonstrated the therapeutic potential of targeting BRD4 in OA, showing that BRD4 inhibition using JQ1 suppressed IL-1β-induced expression of key molecules, including MMPs, pro-inflammatory cytokines that contribute to OA pathology. JQ1 and several small molecular inhibitors of the BET family, including I-BET151 and I-BET762, have been studied for their effects in reducing pro-inflammatory cytokines and their roles in the treatment of inflammatory diseases [28,29]. Emerging evidence has suggested that BET inhibition exerts potent effects by interfering with the core transcriptional program in inflammatory diseases. For example, BRD4 is functionally required for the expression of NF-κB-dependent inflammatory genes, and BRD4 inhibition blocks toll-like receptor 3 (TLR3)-dependent neutrophilia and RSV-induced inflammation by attenuating NF-κB signalling pathway activation. We observed that BRD4 inhibition suppressed IL-1β-induced phosphorylation and translocation of p65 [62]. Consistent with our data, recent studies have shown that BET inhibition suppressed TNF-α-induced NFκB–dependent transcription and NF-κB target genes [28]. These results suggest that BET proteins might be potential therapeutic targets for

cartilage caused by various catabolic factors. On the one hand, these catabolic factors, including aggrecanases and matrix metalloproteinases (MMPs), maintain the dynamic balance of articular cartilage by regulating synthesis and degradation of the extracellular matrix (ECM), which leads to changes in the production and secretion of various proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-ɑ. On the other hand, pro-inflammatory cytokines also potently induce the production of catabolic factors. The two reciprocal function aggravate each other and finally cause the dysregulation of homeostasis in cartilage. The proinflammatory cytokine IL-1β is a critical catabolic factor. Chondrocytes are usually exposed to IL-1β to mimic the microenvironment of OA for vitro research [56]. IL-1β stimulates the synthesis of ECM-degrading enzymes, such as collagenases and aggrecanase, and down-regulated the mRNA levels of Col1a1 in sw1353 cells [57–59]. The results suggesting that sw1353 represent the biologic behavior of human articular chondrocytes [60]. In our study, the primary chondrocyte further support the results of sw1353 cells, and these cells were thus chosen as an in vitro OA model in our study. BRD4 is an important member of the Bromo and Extra-Terminal (BET) domain family, and previous studies have revealed that BRD4 is essential for inducible inflammatory gene expression and plays important therapeutic roles in inflammation. In the study of arthritis and cartilage diseases, many studies have reported the significance of BRD4

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Fig. 8. JQ1 intro-articular injection rescues OA mouse cartilage destruction in vivo. (A) Histopathological evaluation of the articular cartilage of different treatment groups using H & E and Safranin O/Fast Green staining. Scale bar, 800 μm (top), 200 μm (bottom); (B) The overall OARSI histological score was assessed, and a significant decrease in score was observed in the JQ1 intra-articular injection group compared with the PBS injection group. (C) Body weights were recorded twice a week. (D) H & E staining of important organs. Scale bars = 100 μm. (*) P < 0.05, significantly different compared with the model group.

space to activate pro-inflammatory reactions [63]. We further explored the role of BRD4 in mediating transcriptional regulation of HMGB1, and our results demonstrated that IL-1β-induced up-regulation and translocation of HMGB1 from the nucleus to the cytoplasm was dependent on the presence of BRD4 in chondrocytes. Meanwhile, our ChIP results demonstrated that BRD4 regulates the expression of HMGB1 induced by IL-1β partly by directly binding to the HMGB1 upstream non-promoter region, and this process is reversed by JQ1 pre-treatment. Our results further elucidate the transcriptional regulatory mechanism of HMGB1 and increase the therapeutic targets of OA. To further investigate the potential clinical applications of JQ1, we used an ACLT OA model to assess the potential therapeutic effects of intro-articular JQ1 on OA cartilage destruction. We found that JQ1 rescues ACLT OA mouse cartilage destruction in a dose-dependent manner and down-regulates the expression of HMGB1 in cartilage. Thus, BRD4 may be a new target for the treatment of OA.

inflammatory arthritis. BRD4 regulates gene transcription by indirectly recruiting P-TEFb transcription factors to gene promoters [16] and phospho-Ser 2 of the carboxy-terminal domain of RNA polymerase (Pol) II to enhance transcriptional elongation or by directly binding acetylated Lys side chains of modified histones [18]. HMGB1 is an abundant non-histone nuclear protein that is predominantly expressed in the nuclei of eukaryotic cells and plays an important role in the regulation of inflammation. We found that the expression of BRD4 was positively correlated to the expression level of HMGB1, which is one of the most commonly damage associated molecular markers of OA [35]. Notably, JQ1 down-regulated the mRNA and protein levels of HMGB1, and the gene transfection results also revealed that HMGB1 is a direct target of BRD4. A previous study demonstrated that HMGB1 can be either actively or passively released from the nucleus to the cytoplasm following stimulation with pro-inflammatory cytokines and is finally released into the extracellular 3013

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Fig. 9. Schematic of the role of BRD4 in the pathogenesis of OA. BRD4 inhibition attenuates the expression of catabolic genes and pro-inflammatory cytokines during the progression of OA. BRD4 inhibition attenuated NF-κB signalling pathway activation. Furthermore, BRD4 is required for the expression and translocation of HMGB1. Above all, BRD4 inhibition attenuates the degradation of articular cartilage and may be a new promising therapeutic strategy for OA.

In summary, we demonstrated for the first time that BRD4 was upregulated in OA cartilage and was positively correlated with the severity of OA. In addition, as described in Fig. 9, we illustrated the effects of BRD4 inhibition on the expression of catabolic genes and proinflammatory cytkines during the progression of OA in vitro and the effects of JQ1 on the articular cartilage destruction caused by ACLT in vivo. We showed that BRD4 is required for the expression and translocation of HMGB1. BRD4 inhibition attenuated NF-κB signalling pathway activation, possibly through inhibition of transcriptional activity of HMGB1, which requires further exploration. Collectively, our data suggests that BRD4 inhibition may be a new promising therapeutic strategy for OA.

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5. Conclusion We showed for the first time that BRD4 was increased in OA cartilage and was positively correlated with the severity of OA. We further investigated the anti-inflammatory and anti-catabolic effects of JQ1 in chondrocytes. We proposed that HMGB1 is a direct target of BRD4. Additionally, the expression and translocation of HMGB1 was shown to be dependent on the presence of BRD4. We recommend targeting BRD4 as a new strategy for OA treatment, which may be highly efficacious. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbadis.2017.08.009. Conflict of interests All authors declare that they have no conflicts of interest concerning this article. Transparency document The http://dx.doi.org/10.1016/j.bbadis.2017.08.009 with this article can be found, in online version.

associated

Acknowledgements This work was supported by the Shanghai Science and Technology Commission (NO. 13411950503 and NO. 134119a2802), NSFC (NO. 81502604) and the Shanghai Municipal Health and Family Planning Commission (NO. 20164Y0270). References [1] D. Xing, Y. Xu, Q. Liu, Y. Ke, B. Wang, Z. Li, J. Lin, Osteoarthritis and all-cause mortality in worldwide populations: grading the evidence from a meta-analysis, Sci Rep 6 (2016) 24393.

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