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Phosphoproteomics reveals the BRAF-ERK1/2 axis as an important pathogenic signaling node in cartilage degeneration

  • Y. Dong
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • P. Wang
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, No. 100, Kexue Avenue, Zhengzhou, Henan Province, 450001, China
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  • M. Zhang
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • L. Xiao
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • Y. Yang
    Affiliations
    Department of Research Management, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • B. Wang
    Affiliations
    Department of Clinical Microbiology, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • Y. Liu
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • Z. Dai
    Correspondence
    Address correspondence and reprint requests to: J. Zheng and Z. Dai, Department of Orthopedics, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Henan University People’s Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China. Tel.: 86-18539260180.
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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  • J. Zheng
    Correspondence
    Address correspondence and reprint requests to: J. Zheng and Z. Dai, Department of Orthopedics, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Henan University People’s Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China. Tel.: 86-18539260180.
    Affiliations
    Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, No.7, Weiwu Road, Zhengzhou, Henan Province, 450003, China
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Open AccessPublished:September 09, 2022DOI:https://doi.org/10.1016/j.joca.2022.08.003

      Summary

      Objective

      Osteoarthritis (OA) causes gradual cellular alterations, structural anomalies and joint dysfunction. Progressive decline of chondrocyte function plays a vital role on OA pathogenesis. Although protein phosphorylation controls cartilage metabolism, its regulation mechanism in OA remains unclear. Thus, proteomic methods were used to investigate phosphorylation changes in preserved and OA articular cartilage samples, and to explore the intervention targets of phosphorylated kinase.

      Methods

      Preserved (control) and lesioned (OA) cartilage samples from OA cases were assessed by phosphoproteomics. Immobilized metal affinity chromatography was performed for phosphopeptide enrichment. Quantitated phosphosites were comparatively assessed in the cartilage sample pair. Kinase–substrate enrichment analyses were carried out for identifying OA-related kinases. BRAF expression in cartilage tissues was assessed by immunohistochemical staining. The effects of BRAF inhibitor on cartilage degeneration were examined in mouse chondrocytes and OA mouse model.

      Results

      High-sensitivity mass spectrometry-based proteomics revealed 7,471 peptides and 4,375 phosphorylated peptides differing between preserved and lesioned cartilage samples, which represented the significant alteration of kinase hubs and transduction pathways. Phosphoproteomics identified BRAF may be involved in developing OA. BRAF regulated the downstream ERK signaling pathway. In addition, BRAF was upregulated in human OA cartilage as shown by immunohistochemistry. Remarkably, BRAF inhibition alleviated cartilage degradation in a mouse model of OA through its downstream of ERK pathway activation.

      Conclusions

      Jointly, these findings provide an overview of phosphoproteomic alterations occurring during cartilage degeneration, identifying the BRAF-ERK1/2 Axis signaling as a potential signaling pathway involved in OA.

      Keywords

      Introduction

      Osteoarthritis (OA) represents the commonest joint pathology, mostly affecting the hips and knees, and characterized by joint pain and dysfunction
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      , its comprehensive study would be very desirable.
      Protein phosphorylation is important post-translational modification (PTM), resulting in structural alterations by affecting the activities of enzymes, protein–protein interactions, and protein functions and cellular localizations
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      . Dysregulated protein phosphorylation in inflammatory response pathways may be involved in development of OA, and our previous study has demonstrated that increased amounts of phosphorylated ERK1/2, JNK and p38, and MAPK pathway activation contribute to cartilage degeneration
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      . The involvement mechanisms for MAPK pathway activation are remained to be elucidated, and the measurement of total proteome and phosphoproteome may be effective methods to identify the potential mechanism of MAPK pathway activation. One study demonstrated that phosphoproteomic analysis of articular synoviocytes was only focused on differential expression of the highly phosphorylated annexin VII (Anx7) protein between RA and OA
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      . Another study utilized quantitative phosphoproteomic analysis to reveal the glycogen synthase kinase3 (GSK3) which plays a vital role in FGF inhibitory response in chondrocytes
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      . Although limited studies have indirectly indicated that phosphoproteomic analysis may aid to explore the potential mechanism of OA, there is still urgent to use the phosphoproteomic analysis to screen the key protein phosphorylation and signaling pathways involved in OA. Therefore, phosphoproteome analysis was carried out to assess phosphorylation alterations between preserved and OA cartilage specimens in humans by using high-resolution MS method. The phosphoproteomics analysis revealed BRAF signaling as a potential signaling pathway in cartilage degeneration, and the regulation potential mechanism was further explored in the anterior crucial ligament transection (ACLT) mouse model as well as in human OA.

      Materials and methods

      Samples

      Preserved (control) and lesioned (OA) cartilage specimens were from OA cases administered joint replacement surgery with end-stage disease. Surgical operations were carried out at the Henan Provincial People's Hospital (Zhengzhou, China). This trial had approval from the Medical Ethics Committee of Henan Provincial People's Hospital and each patient provided informed consent before enrolment. The histological staining was used the HE and Safranin O-Fast green staining. The International Cartilage Repair Society (ICRS) score was used to evaluate the cartilage damage of the two groups which can distinguish preserved and lesioned (OA) cartilage tissue
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      RNA sequencing data integration reveals an miRNA interactome of osteoarthritis cartilage.
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      • Steinberg J.
      • Tuerlings M.
      • de Almeida R.C.
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      • Swift D.
      • et al.
      A molecular map of long non-coding RNA expression, isoform switching and alternative splicing in osteoarthritis.
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      • Ramos Y.F.M.
      • den Hollander W.
      • Bomer N.
      • Nelissen R.
      • Bovee J.
      • et al.
      Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.
      . Cartilage specimens from eight cases were examined (online supplementary Table-S1). The average ICRS score of control cartilage tissue was 1.2, which suggested a mild degeneration of control cartilage.

      Sample preparation for phosphoproteomics

      Cartilage tissues underwent lysis with four volumes of lysis buffer with 8M urea by sonication. After centrifugation at 12,000 g at 4°C (10 min), the precipitate added to a clean tube was used for supernatant phosphoproteomics analysis. Protein quantitation utilized the BCA kit as directed by the manufacturer. Totally 800 μg protein underwent successive incubations with 5mM dithiothreitol (30 min at 56°C) and 11mM iodoacetamide (15 min at ambient) shielded in darkness. Then, the specimens were diluted with 100mM TEAB, digested with trypsin at 1/50 (w/w) overnight. Phosphopeptide enrichment was performed with a Fe3+ IMAC resin (Thermo Fisher Scientific, China) in this study according to a routine protocol
      • Wu C.T.
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      • Xiao Y.
      • Lee I.T.
      • Cheng R.
      • Nakayama T.
      • et al.
      SARS-CoV-2 infects human pancreatic beta cells and elicits beta cell impairment.
      . Briefly, prior to 500 μg peptide mixture underwent incubation with IMAC microspheres, which were collected via centrifugation and submitted to two washes with 50% acetonitrile and 6% trifluoroacetic acid, the peptides were cleaned with C18 ziptips (millipore), according to the manufacturer's instructions. Then, the enriched phosphopeptides were eluted under shaking conditions with elution buffer containing 10% NH4OH.

      Mass spectrometry

      The peptide segments were dissolved by liquid chromatography mobile phase A and separated by nanoelute ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile. Mobile phase B is a solution containing 0.1% formic acid and 100% acetonitrile. The gradient elution procedure with a flow rate at 300 nl/min by using different volume ratio of solvents in a binary solvent mixture as follows: 7%–22% B (0–40 min), 22%–30%B (40–52 min), 30%–80%B (52–56 min), 80%B (56–59 min). Peptide separation utilized UPLC system, which was followed by injection into a capillary ion source for ionization, then analyzed by timsTOF Pro mass spectrometry (single-run proteome and phosphoproteome analysis respectively). The ion source voltage was set at 1.75 kV, and the peptide parent ion and its secondary fragments were detected and analyzed by high-resolution TOF. detector. The scanning range of secondary MS is set to 100–1700 Da. Data were acquired in the PASEF mode. After primary mass spectra were obtained, secondary spectra with the charge numbers of respective parent ions of 0–5 were obtained for 10 times. The dynamic exclusion time of tandem MS scanning was set to 30 s to avoid the repeated scanning of the parent ion
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      • et al.
      SARS-CoV-2 infects human pancreatic beta cells and elicits beta cell impairment.
      .

      Phosphoproteome data processing

      Raw MS data were analyzed with MaxQuant v1.5.2.8. MS/MS data were assessed by matching them based on the human UniProt database derived from the fasta file of Homo_sapiens_9606_SP_20190513 (FDR<0.01; enzyme specificity, trypsin; ≤4 missing cleavages allowed). Mass tolerance for precursor ions was 20 and 5 ppm in the initial and main searches, respectively, vs 0.02 Da for fragment ions. For phosphopeptide identification, a minimum score threshold of 40 was used. Phosphosites with more than 0.75 localization probability were used for subsequent analysis. In addition to carbamidomethyl on Cys was specified as fixed modification, acetylation on protein N-terminal, oxidation on Met, and phosphorylation on Ser, Thr, Tyr were specified as variable modifications. All MS data, including annotated mass spectra, have been deposited to the Proteome X change Consortium via the PRIDE partner repository (data set no. PXD030425).

      Phosphoproteome bioinformatics data analysis

      The normalization of phosphoproteome intensities for each sample was obtained by subtracting the corresponding median intensities. Phosphoproteome intensities underwent normalization by subtracting the median intensities of each sample. The phosphorylation sites identified in more than two samples of each group were used for subsequent analysis. Proteins were annotated for categories in the R software as GO's biological process (BP), molecular function (MF) and cellular component (CC) as well as KEGG pathways. Kinase activity prediction analysis was performed with the iGPS software.

      Animal study

      Twelve-week-old specific pathogen-free (SPF) male C57BL/6 mice (20–25 g) underwent housing at the Experimental Animal Center of the People's Hospital of Zhengzhou University, and were fed rodent chow. The ACLT surgery of the right knee joint was performed for inducing experimental OA in mice, based on a previous report
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      • Blanchet T.J.
      • Morris E.A.
      The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.
      . Based on the random number table, totally 40 mice were randomized into the sham (sham animals treated with dimethyl sulfoxide as vehicle), sham + BRAF inhibitor (sham-operated mice treated with a BRAF inhibitor: B-Raf inhibitor 1(Compound 13 (Selleck, China) administration at 200 μg/kg), ACLT (ACLT surgery and vehicle administration) and ACLT + BRAF inhibitor (ACLT surgery and BRAF inhibitor administration at 200 μg/kg) groups. The above-mentioned each group mice (n = 10) were further separately allocated to two cages by randomization to keep enough activity space for mouse. Intra-articular injections of vehicle and BRAF inhibitor, respectively, were carried out twice weekly for 4 weeks in total. Experiments involving animals were approved by the Medical Ethics Committee of Henan Provincial People's Hospital.

      Histology and immunochemistry

      Human cartilage and mouse joint tissue specimens underwent fixation with 4% paraformaldehyde (Sigma–Aldrich, USA) for 2 days. After decalcification and paraffin embedding, sectioning was performed at 4-μm. This was followed by hematoxylin-eosin (HE (Sigma–Aldrich, USA)) and Safranin O-Fast green ((Sigma–Aldrich, USA). The ICRS cartilage lesion classification system was used to assess the human OA samples
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      . The destruction of mouse articular cartilage was evaluated by the Osteoarthritis Research Society International (OARSI) scoring system in a double blinded manner
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      The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.
      . Briefly, the tissue specimens from human cartilage and mouse were cut into 4 μm sections in a sagittal orientation and then three 4 μm sections were placed on each slide. We used 12 slides for histologic scoring of the entire articular surface. Immunohistochemical staining used the DAB tissue staining SP-kit for development. Specimens were successively incubated with normal horse serum for 20 min, primary anti-BRAF and anti-MMP-13 (Abcam, USA) antibodies has been used in previous studies
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      overnight at 4°C and appropriate secondar antibodies for1 h at ambient. Hematoxylin counterstaining was carried out before optical microscopy. The rates of BRAF and MMP-13 positive cells of this study were determined using Image-Pro Plus 6.0 (Media Cybernetics, USA), and calculated as the number of BRAF or MMP-13 positive chondrocytes divided by the total number of chondrocytes in each section.

      Cell experiments

      Several studies have indicated that chondrocytes were isolated from femoral condyles and tibial plateaus of 3–8 day old mice which exhibited cartilage properties
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      Regulation of the catabolic cascade in osteoarthritis by the zinc-ZIP8-MTF1 axis.
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      Kdm6b regulates cartilage development and homeostasis through anabolic metabolism.
      . In vitro study, primary chondrocytes were derived from knee joint of 5-day-old C57/BL mouse. All the experiments were approved by the Medical Ethics Committee of Henan Provincial People's Hospital before this study was conducted. Mouse cartilages from 5-day-old mice were minced in chilled phosphate buffer saline, followed by 0.25% trypsin (Gibco, USA) digestion at 37°C (30 min) and incubation with 0.25% collagenase II (Gibco, USA) at 37°C for 24 h. Chondrocyte culture was carried out in DMEM/F12 containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma–Aldrich, USA) at 37°C in a 5% CO2 incubator. Cells underwent two passages before utilization in assays.
      To evaluate the response of chondrocytes under inflammatory environment in vitro. passage-3 chondrocytes were seeded in 6-well plates at 2 × 105/well, and we examined the expression of BRAF, ERK1/2, and p-ERK1/2 in chondrocytes that had been stimulated with IL-1β for 0, 15, 30 and 60min with or without BRAF inhibitor.

      Quantitative real-time PCR (qRT-PCR)

      Mouse chondrocyte seeding was carried out in 6-well plates with DMEM (Gibco, USA) containing 10% FBS. Cells were administered IL-1β (10 ng/mL) (PeproTech, USA) for 3 days with or without B-Raf inhibitor 1 (5μM). Total RNA was extracted from chondrocytes by using TRIzol® Reagent (Invitrogen, USA). Reverse transcription involved 1 μg RNA was carried out with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). MMP-13, Collagen X, Collagen II and aggrecan mRNA amounts were assessed by qRT-PCR using specific primers (online supplementary Table- S2). GAPDH was utilized for normalization in the 2−ΔΔCt method.

      Western blotting

      The mouse primary chondrocyte protein extracts were obtained using RIPA Buffer containing protease and phosphatase inhibitors (Boster BIO, China). 20 μg of total protein underwent separation by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by transfer onto PVDF membranes (Millipore, USA) and a 1-h blocking with 5% fat free milk in Tris-buffered saline containing 0.1% Tween-20. Then, successive incubations were performed with rabbit anti-BRAF (1:1000), ERK1/2 (1:1000) p-ERK1/2 (1:1000) and β-actin (1:1000) (Cell Signaling, MA, USA) primary for overnight at 4°C and anti-rabbit secondary (Cell Signaling, MA, USA) for 1 h at ambient antibodies. Enhanced chemiluminescence kit (Bio-Rad, USA) was utilized for detection.

      Statistical analysis

      Kolmogorov–Smirnov test was used to assess normal distribution of the data before the data analyses. Normally distributed measurement data are presented as the mean ± standard deviation (SD). Pearson's correlation analysis was used to assess correlation of each paired preserved regions, lesioned regions or preserved and lesioned regions. A Welch t-test was utilized for identifying phosphosites in preserved and lesioned group. The Fisher's exact test was performed for comparisons of significant enrichment between two groups. Benjamini and Hochberg FDR was utilized to correct for multiple comparisons. Comparisons between two groups of ICRS scores and quantification of BRAF-positive cells measurements were analyzed by independent-sample t-tests. Relative mRNA amounts, OARSI scores and MMP-13-positive cells comparison between multiple groups were performed by one-way analysis of variance. All data analysis was performed in SPSS 20.0 software (IBM, USA). A value of P < 0.05 was considered as a statistically significant difference.

      Results

      Phosphoproteomic status in OA patient cartilage

      The phosphorylation of signaling effectors and downstream targets in the cartilage are known to be involved in the pathological process of OA. However, the overall phosphorylation profile in OA cartilage has not been assessed by phosphoproteomics. We developed a phosphoproteomic procedure to assess preserved and lesioned OA cartilage specimens [Fig. 1(A)]. Table S1 lists patient features. The differences in the phosphorylation levels of proteins were examined by the label-free LC−MS/MS technique.
      Fig. 1
      Fig. 1Experimental approach of the current study: A. Experimental workflow used to analyze the phosphoproteome of preserved and lesioned regions in human OA cartilage. B. Number of identified proteins, quantifiable proteins, and quantifiable sites. C. Proportions of phosphoserine, phosphothreonine and phosphotyrosine sites. D. Different expression of phosphorylated proteins data from human cartilage (control, red; OA, green) were assessed by principal component analysis, and analysis was performed after intensity normalization in various channels. E. Correlation matrix of phosphoproteomic data of each paired preserved regions, lesioned regions or preserved and lesioned regions were represented by using heat map.

      Quantitative signatures of preserved and lesioned areas of OA cartilage

      To assess whether the preserved and lesioned regions of the cartilage correspond to phosphor-signaling in OA chondrocytes, phosphoproteomics was carried out, and the quantitative phosphorylation sites were selected by using t-test, that is, at least two of them were identified in OA and control groups. Totally 7,471 peptides and 4,375 phosphorylated peptides were detected based on MS. In addition, 6,146 phosphorylation sites were identified in 2,207 proteins, including 2,858 sites in 1438 proteins with quantitative data [Fig. 1(B)]. The 2,858 phosphosites included 2,549 phosphoserine (89%), 281 phosphothreonine (10%) and 28 phosphotyrosine (1%) sites [Fig. 1(C)]. These quantitative data were assessed for pathway enrichment and kinome profiling. First, PCA was carried out for clearly classifying phosphoproteomic data of the preserved and lesioned regions. PCA shows a good clustering among repeated specimens, indicating good quantitative repeatability [Fig. 1(D)]. The results from Pearson's correlation analysis shows that correlation coefficients between each paired of preserved regions, lesioned regions or preserved and lesioned regions were all over than 0.8, indicated that strong positive correlations between all combinations of samples [Fig. 1(E)].
      Then, phosphosites exhibiting statistical difference between preserved and lesioned tissue specimens were further examined. All phosphosites were shown in the Supplementary materials (phosphosites identified information). The Welch t-test adjusted by permutation test was utilized for identifying 154 phosphosites elevated in OA lesioned tissue samples and 258 reduced in OA lesioned tissues.

      Differential expression of phosphorylated modified proteins in OA cartilage

      Next, protein phosphorylation levels in preserved and lesioned regions in OA cartilage were measured to identify the signaling pathway and its key regulation proteins. In lesioned regions, as shown in Fig. 2, the cytoskeletal regulation proteins were downregulated in lesioned regions such as CFL1, VCL, SLC9A3R1, SYNM, EPB41L2, NEBL, SVIL, ANXA2 and PRKCA et al. The phosphorylation levels of epigenetic regulatory proteins were decreased, including UBXN7, HIST1H1E, RING1, TRIM28 and HIST2H3A. Cell proliferation related proteins, including TPD52L2, LGALS1 and YAP1, showed decreased phosphorylation levels. The phosphorylation levels of collagen-synthesis proteins and hyaluronic acid-synthesis proteins were reduced, including ECM2, HAPLN1, BGN and FMOD, whereas the phosphorylation levels of type I collagen formation and biosynthesis proteins (FN1 and VIM) were increased. The phosphorylation levels of lipid synthesis and transport proteins were increased, including APOL1, ANXA2 and FNDC3B. Similarly, some pathways related to inflammation were significantly enriched. The phosphorylation levels of SIRT1 were decreased, SIRT1 amounts are decreased in multiple chronic inflammatory pathologies
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      . Meanwhile, the phosphorylation levels of PEBP1 were reduced. PEBP1 suppresses RAF1's kinase activity via dissociation of the RAF1/MEK complex
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      .
      Fig. 2
      Fig. 2Phosphoproteomic profiles of preserved and lesioned regions in human OA cartilage. Expression patterns shown by the heat map after unsupervised hierarchical clustering of differentially phosphorylated proteins.

      Classification based on gene ontology (GO) annotation

      Furthermore, GO-based annotation was used to categorize the proteins based on their CC, MF and BPes. For CC, the proteins were mainly involved in extracellular region, extracellular region part, extracellular vesicle, extracellular organelle and extracellular exosome. For MF, the proteins were mainly associated with cytoskeletal protein binding, structural molecule activity and tubulin binding. For BPes, the majority of enriched categories were associated with actin cytoskeleton organization, actin filament organization, regulation of cellular response to stress and regulation of cell size (Fig. 3).
      Fig. 3
      Fig. 3GO terms with significant enrichment involving proteins showing phosphorylation sites in human OA cartilage. Lesioned and preserved regions of the cartilage were assessed for phosphorylation sites. The Fisher's exact test was performed for comparisons different in proteins expression between lesioned and preserved regions and furthermore Benjamini and Hochberg FDR was utilized to correct for multiple comparisons.

      OA-specific kinome profiling of OA cartilage

      Identifying druggable active kinases may guide anti-OA treatment, and could be achieved by phosphoproteomic analysis of preserved and lesioned OA cartilage specimens. Phosphosites on protein kinases were counted based on phosphoproteomic data (Fig. 4). We screened five kinases that may play a role in OA, namely: YANK1, KSR1, ARAF, KSR2 and BRAF.
      Fig. 4
      Fig. 4A. Kinome profiles based on phosphoproteomic data of preserved and lesioned regions in OA cartilage. Kinases with statistically significant differences are depicted. Kinases were predicted to be activated (red) or inactivated (blue) in OA cartilage. B. NES is the normalized enriment score of predicted kinase activity by using the iGPS software. The picture showed the PPI network information of BRAF and its interacted with proteins.

      BRAF is upregulated in OA cartilage

      We harvested cartilage samples from patients, and performed HE and Safranin O-Fast green staining. ICRS scores were used to confirm the difference of moderate and severe degenerated cartilage (Fig. 5). We measured BRAF levels in preserved and OA cartilage specimens by immunohistochemistry. Preserved cartilage showed low BRAF-positive cell rates in all zones. In contrast, in OA cartilage, chondrocytes showed strong expression of BRAF in the middle and deep zones. BRAF-positive cell rates were significantly increased in OA cartilage specimens compared with preserved cartilage samples using a double-blinded manner (Fig. 5).
      Fig. 5
      Fig. 5BRAF is overexpressed in human osteoarthritis (OA) cartilage. A. Hematoxylin and eosin, Safranin O-fast green and immunohistochemical staining for BRAF in human cartilage. Scale bar = 100 μm. B. ICRS (International Cartilage Repair Society) scores and quantification of BRAF-positive cells in control and OA human cartilage specimens. Date are means ± SD (n = 8; ∗p < 0.05).

      BRAF inhibition alleviates cartilage degradation in the mouse model of OA

      Next, BRAF's role in OA progression was assessed in vivo, using a BRAF suppressor (B-Raf inhibitor 1) administered via intra-articular injection in the ACLT-induced mouse model of OA. Safranin O and fast green staining demonstrated cartilage surface improvement in the ACLT mouse model following B-Raf inhibitor 1 administration [Fig. 6(A)]. Quantitation by OARSI scoring demonstrated B-Raf inhibitor 1 significantly lowered OARSI scores [Fig. 6(B)]. In addition, ACLT-related MMP-13 upregulation was decreased [Fig. 6(C)].
      Fig. 6
      Fig. 6BRAF inhibition alleviated osteoarthritis in mice (A) HE and Safranin O-Fast green staining of mouse knee joint sections. Immunostaining of MMP13 in each group joint sections. (B) OARSI histological scores in each group joint sections (C) Quantification of MMP13-positive cells in each group joint sections. Date are means ± SD (n = 10; ∗p < 0.05).

      BRAF suppression reduces IL-1β-induced chondrocyte catabolism

      The effect of B-Raf inhibitor 1 on IL-1β-induced chondrocyte degeneration was measured. After mouse chondrocytes were treated with B-Raf inhibitor 1 and IL-1β for 72 h. The upregulation expression of collagen X and MMP-13 mRNA expression were observed, whereas downregulation of collagen II and aggrecan mRNA expression were observed, after the cells were treated with IL-1β. B-Raf inhibitor 1 can partly rescue the effect of cells induced by IL-1β [Fig. 7(C)].
      Fig. 7
      Fig. 7BRAF suppression alleviates IL-1β-induced chondrocyte catabolism. A. Chondrocytes were administered IL-1β (10 ng/mL) at various times. β-actin, ERK1/2, p-ERK1/2 and BRAF protein amounts were assessed by immunoblot. B. Chondrocytes underwent a 16-h culture in medium with no serum, pretreatment with a BRAF inhibitor for 24 h, and induction with IL-1β (10 ng/mL) for 60 min. β-actin, ERK1/2, p-ERK1/2 and BRAF protein amounts were assessed by immunoblot. C. Relative mRNA amounts of MMP-13, Collagen X, Collagen II and Aggrecan in chondrocytes. Data are means ± SD from three technical replicates. ∗p < 0.05.
      BRAF-ERK1/2 pathway activity in IL-1β-induced chondrocytes was also assessed. As shown by immunoblot, BRAF and phosphorylated ERK1/2 (p-ERK1/2) amounts were elevated upon IL-1β stimulation [Fig. 7(A)], and B-Raf inhibitor 1 attenuated these effects [Fig. 7(B)].

      Discussion

      This work assessed the phosphoproteomic profiles of paired preserved and lesioned OA cartilage specimens, presenting the first comprehensive, OA-specific protein phosphorylation map. Totally 2,858 phosphorylation sites were identified on 1438 proteins in cartilage samples. This study found strong positive correlations between all combinations of samples. We hypothesized the reasons as follows: first, both the preserved and lesioned cartilage specimens were from OA cases administered joint replacement surgery with end-stage disease which may cause no substantial changed in protein expression between preserved and lesioned cartilage specimens. Second, this study was only identified 1438 proteins existed in human cartilage specimens between preserved and lesioned cartilage specimens, which may reduce its chance to reveal the difference of protein expression between preserved and lesioned cartilage specimens. Third, owing to the protein expression in the cartilage specimens was quite low, the endogenous and exogenous factors may be failed to induce difference in protein expression between preserved and lesioned cartilage specimens.
      The data confirmed well-known features of cartilage degeneration and revealed multiple kinases activated in lesioned OA cartilage tissue samples, which might constitute new therapeutic targets. We screened BRAF, YANK1, KSR1, ARAF and KSR2 kinases through kinase prediction. The upregulation of BRAF protein expression in OA cartilage was verified; BRAF inhibition alleviated cartilage degradation in the mouse OA model. Further investigation demonstrated the ERK1/2 pathway is a downstream gene of BRAF in OA. Taken together, these data suggest that protein phosphorylation collectively affects cartilage degeneration, exemplifying the complexity of functional validation of protein kinase–protein networks.
      It is well known that IL-1β, as an important inflammatory factor involved in the occurrence of human osteoarthritis, is increased secreted in the joint fluid of patients with knee osteoarthritis. In vitro study, IL-1β (10 ng/mL) is widely used to induce the inflammatory environment of osteoarthritis. Evidence indicated that altered cartilage phenotype of chondrocytes were observed after chondrocytes were treated with IL-1β for 72 h
      • Laiguillon M.C.
      • Courties A.
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      • Auclair M.
      • Sautet A.
      • Capeau J.
      • et al.
      Characterization of diabetic osteoarthritic cartilage and role of high glucose environment on chondrocyte activation: toward pathophysiological delineation of diabetes mellitus-related osteoarthritis.
      ,
      • Thompson C.L.
      • Patel R.
      • Kelly T.A.
      • Wann A.K.
      • Hung C.T.
      • Chapple J.P.
      • et al.
      Hedgehog signalling does not stimulate cartilage catabolism and is inhibited by Interleukin-1beta.
      . The current data showed that total BRAF and p-ERK1/2 was upregulated induced by IL-1β stimulation. An BRAF inhibitor could alleviate IL-1β′s effect on p-ERK. BRAF inhibition also attenuated IL-1β-mediated Collagen X and MMP-13 upregulation, and Collagen II and Aggrecan downregulation at the mRNA level. More recently it was shown that Wnt-3a induces the phosphorylation of BRAF by CaMKIIα and ERK1/2 signaling activation, which results in chondrocyte de-differentiation
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      Non-canonical Wnt induces chondrocyte de-differentiation through Frizzled 6 and DVL-2/B-raf/CaMKIIalpha/syndecan 4 axis.
      . All these data indicate BRAF-ERK1/2 signaling is important in cartilage degeneration and differentiation. ERK1/2, induced by MKK1 and MKK2, respectively, constitute one of the subfamilies of MAP kinases that are broadly produced by many cell types (e.g., joint tissues) and most frequently detected together
      • Rudolph J.
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      Slow inhibition and conformation selective properties of extracellular signal-regulated kinase 1 and 2 inhibitors.
      . Besides modulating joint degeneration, ERK1/2 may control pain signals
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      Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1beta-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways.
      . Non-joint studies also indicated a role for ERK in pain signaling
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      .
      The finding of phosphorylation levels of CFL1 and VCL reduction in OA patients indicated that cytoskeletal remodeling may relate to cartilage degeneration. CFL1 can regulate mesenchymal stem cells (MSCs) towards to the chondrogenic lineage
      • Ohashi K.
      Roles of cofilin in development and its mechanisms of regulation.
      ,
      • Borovac J.
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      Regulation of actin dynamics during structural plasticity of dendritic spines: signaling messengers and actin-binding proteins.
      . CFL1 induces the dissociation of actin microfilaments, altering the mechanical features and extracellular matrix (ECM) assembly of cells
      • Tay L.X.
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      Differential protein expression between chondrogenic differentiated MSCs, undifferentiated MSCs and adult chondrocytes derived from Oryctolagus cuniculus in vitro.
      . VCL as an adhesion protein, not only links the ECM and the actin cytoskeleton, and also relates to chondrocyte differentiation. For instance, a certain degree of vibration induces chondrocytes VCL mRNA up-regulated expression
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      Beneficial effects of low frequency vibration on human chondrocytes in vitro.
      . Taken together, both CFL1 and VCL, related to the cytoskeleton which might contributed to the pathological process of OA by regulating of chondrocyte differentiation, and altering their phosphorylation state.
      Furthermore, PEBP1 and SIRT1 were de-phosphorylated in lesioned OA cartilage tissues contributing to the pathological process of osteoarthritis. PEBP1 is also referred to as Raf kinase inhibitor protein (RKIP). RKIP is indeed a broadly expressed suppressor of Raf-MEK-ERK signaling
      • Rath O.
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      • et al.
      The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.
      . Evidence have demonstrated RKIP can suppress MEK phosphorylation by Raf-1 and exert its role by controlling the generation of a kinase–substrate complex through direct interaction with both Raf-1 and MEK
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      Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP.
      . Whilst studying mandibular dysplasia, researchers found physiological mechanical stimulation induces cartilage growth alteration by increasing RKIP amounts via inhibition of ERK signaling
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      Mechanical stress promotes matrix synthesis of mandibular condylar cartilage via the RKIP-ERK pathway.
      . SIRT1, a NAD-dependent deacetylase, is involved in anabolism in the cartilage
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      The protective mechanism of SIRT1 on cartilage through regulation of LEF-1.
      . SIRT1 is indispensable in chondrogenic differentiation of MSCs, and its expression in articular cartilage is inversely correlated with knee OA severity
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      Sirtuin-1 (SIRT1) is required for promoting chondrogenic differentiation of mesenchymal stem cells.
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      The expression of SIRT1 in articular cartilage of patients with knee osteoarthritis and its correlation with disease severity.
      . SIRT1 upregulation also suppresses OA chondrocyte apoptosis and ECM degeneration by upregulating Bcl-2 and downregulating Bax, MMP 1 and MMP 13 via reduction of phosphorylated p38, JNK and ERK levels
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      . As mentioned above, dephosphorylation of PEBP1 and SIRT1 may be as key regulation proteins to modulate the signaling pathways involved in the development of OA.
      Additionally, ANXA2 and APOL1 phosphorylation levels were up-regulated in lesioned OA cartilage tissues. ANXA2 is a positive regulator of low-density lipoprotein receptors, whose amounts and phosphorylation levels are increased in synovial tissue specimens from individuals with rheumatoid arthritis
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      The protein-protein interaction between connective tissue growth factor and annexin A2 is relevant to pannus formation in rheumatoid arthritis.
      . ANXA2 was identified as a DDR-2 binding protein. It might be phosphorylated to promote MMP-13 secretion, which induces cartilage degeneration
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      The discoidin domain receptor 2/annexin A2/matrix metalloproteinase 13 loop promotes joint destruction in arthritis through promoting migration and invasion of fibroblast-like synoviocytes.
      . APOL1 plays an important role in lipid exchange and transport in the whole body, and may contribute to reverse cholesterol transport from peripheral blood cells to the liver
      • Shukha K.
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      Most ApoL1 is secreted by the liver.
      . Meanwhile, investigators found that APOL1 mRNA expression is increased in OA samples
      • Okabe T.
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      . In a word, ANXA2 and APOL1 as the key regulation protein of lipid metabolism may be involved in cartilage degeneration of OA.
      This study firstly performed phosphoproteomics of paired preserved and lesioned OA cartilage specimens. However, additional hurdles need to be overcome before applying the current findings. First, phosphoproteins were the focus of this work, and multiple unphosphorylated proteins are found in cartilage degeneration. Additionally, cartilage samples were mainly from female donors, and male specimens should be comparatively examined. Second, the ACLT mice model was used to assess the potential mechanisms of developing OA in this study which may induce surgical destabilization, however, the ACLT mice model has been confirmed to be suited to imitate the pathogenesis of human OA
      • Yang S.
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      Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction.
      . Third, although an isotype control was not conducted in this study, BRAF antibody was purchased from Abcam company (cat: ab33899), and the specificity of the antibody has been validated in several published literatures
      • Wang X.
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      Conformation-specific effects of Raf kinase inhibitors.
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      Structure-based design and synthesis of 2,4-diaminopyrimidines as EGFR L858R/T790M selective inhibitors for NSCLC.
      • Brown W.S.
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      • Lizee G.
      • McIntyre B.W.
      B-Raf regulation of integrin alpha4beta1-mediated resistance to shear stress through changes in cell spreading and cytoskeletal association in T cells.
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      c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma.
      . Fourth, although a positive control was not conducted, MMP-13 protein expression in the ACLT + BRAF inhibitor group was reduced relative than that of ACLT group at the same cultured conditions indicated that BRAF inhibitor has played partial role in cartilage degeneration at a certain content. Fifth, considering that surgical treatment may lead to one cage of mice more active than another cage, all groups were performed intra-articular injections after 1 week post-operation to keep the similarity-activity of mice between control sham and surgical groups. Finally, we performed phosphorylated proteomic analysis on cartilage tissue samples, while OA is a whole joint disease, also affecting synovial inflammatory response, osteophyte formation, and subchondral bone remodeling.
      Complex pathologies scarcely result from abnormalities in a single gene, but rather from alterations of global cell networks. In summary, a comprehensive overview of the dynamic phosphorylation changes in OA cartilage was provided. Integrating these large-scale data with previously reported signaling pathways may help understand how protein phosphorylation and signaling events modulate cartilage degeneration. Hyperactivation of the kinase BRAF in OA was remarkable, with a prominent regulatory function in cartilage degeneration.
      In conclusion, taken together, the present phosphoproteomic and functional data support a model whereby an OA environment, including cartilage degeneration, leads to OA at least in part through the BRAF-ERK1/2 axis. These findings provide a molecular basis for application of BRAF inhibitor to treatment OA. Further related in-depth studies may be needed to explore application of BRAF inhibitor to treatment OA patients.

      Ethic statement

      The manuscript “Phosphoproteomics Reveals the BRAF-ERK1/2 Axis is an Important Pathogenic Signaling Node in Cartilage Degeneration”, which was written by Yonghui Dong et al., has been reviewed by Medical Ethics Committee of Henan Provincial People's Hospital. The content and process of the manuscript conform to the ethical requirements of biomedical research promulgated by the international and national governments and we agree to submit the article.

      Authors' contributions

      YHD drafted the work. JZ and ZPD designed the experiments. PW and BYW collected the samples. YGY and YHD analyzed the data. YKL and MZ interpreted the results. JZ and ZPD revised the work. All authors read and approved the final manuscript.

      Acknowledgements

      Not applicable.

      Conflict of interest

      The authors declare that they have no conflicts of interests.

      Funding

      This study is supported by Henan Provincial Natural Science Foundation of China ( 212300410242 ), National Natural Science Foundation Youth Project of China (82002300), and Henan Young and Middle-aged Health Science and Technology Innovation Excellent Youth Talent Training Project of China (YXKC2021047).

      Availability of data and materials

      All data generated or analyzed during this study are included in this published article and its supplementary information files.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:

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