Changes in the epigenetic status of the SOX-9 promoter in human osteoarthritic cartilage

Authors


Abstract

Whether osteoarthritis (OA) is associated with alterations in the epigenetic status of anabolic factors is largely unknown. To answer the question, we investigated the DNA methylation and histone modification of SOX-9 gene promoter, a typical anabolic gene, in the articular cartilage from nine patients with femoral neck fractures without OA and from nine hip OA patients. Methylation-specific PCR (MSP) and bisulfite sequencing analysis (BSQ) showed that the methylation of SOX-9 promoter was increased in OA cartilage compared to normal cartilage. The decreased SOX-9 gene and protein expression in OA chondrocytes was reversed by the treatment of 5-azacytidine (5-AzaC), the demethylating agent. Methylation of SOX-9 proximal promoters reduced the binding affinity of transcription factors CCAAT-binding factor/nuclear factor-Y and cyclic adenosine monophosphate (cAMP) response element–binding. There was a significant increase in H3K9 and H3K27 trimethylation and a significant decrease in the acetylation of H3K9, 15, 18, 23, and 27 at SOX-9 promoters in OA chondrocytes. These findings suggest that hip OA is associated with a change in the epigenetic status of SOX-9 promoter, including increased DNA methylation and altered histone modification. © 2013 American Society for Bone and Mineral Research.

Introduction

Osteoarthritis (OA) is a common cause of disability in the elderly persons.1 Gradual degeneration of articular cartilage (AC) leads to joint pain and dysfunction in OA.2 Although known risk factors comprise age, obesity, and abnormal joint loading, OA is now understood to be more than just the result of “wear and tear.”3, 4 A deregulation in balance between anabolic and catabolic mechanisms maintaining the extracellular matrix (ECM) plays a key role in the pathogenesis of OA.5, 6 The associated cellular metabolic changes are epitomized by downregulation of anabolic genes, including transforming growth factor (TGF)-βs, insulin-like growth factor (IGF)-1, SOX trio, type II collagen, aggrecan, and upregulation of catabolic genes such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs).7

Recently, epigenetics has been spotlighted as an important mechanism for gene regulation.8 Genomic DNA methylation and modification of nucleosomal histone tails are essential epigenetic changes that control the chromosome remodeling.9, 10 DNA methylation is the addition of a methyl group to the 5′ cytosine in a CpG dinucleotide, which favors genomic integrity and largely contributes to gene silencing.11 Genomic DNA methylation is the most fundamental process that leads to gene silencing.12, 13 Genomic DNA methylation occurs as a heritable modification during cellular replication and lineage differentiation, playing a crucial role in early embryonic development.14–17

Although the changes in the methylation of oncogenesis-related genes have been extensively studied,18–20 a limited number of studies investigated the altered methylation in OA.4, 7, 21–25 Roach and colleagues7 investigated the methylation status of the promoter regions of MMP3, MMP9, MMP13, and ADAMTS-4. The overall percentage of nonmethylated sites in the promoter site of these enzymes increased in the OA cartilage.7 DNA hypomethylation status correlated with mRNA and protein expression of ADAMTS-4 by OA chondrocytes.21 Methylation of leptin gene decreased in OA chondrocytes, and downregulation of leptin with small interference RNA (siRNA) dramatically inhibited MMP-13 expression.26 On the other hand, there was no significant correlation between aggrecan mRNA expression levels and methylation status in normal (aged) and OA chondrocytes.4 In addition, no difference was found in methylation of the promoter of the p21 (WAF1/CIP1) gene that was significantly downregulated in OA chondrocytes.24 Whereas these studies mostly investigated demethylation of catabolic gene promoters or matrix components in OA, fewer studies have been reported on the DNA methylation of anabolic factors in OA pathogenesis. Positive correlation was reported between age and bone morphogenetic protein (BMP)-7 methylation status in the chondrocytes isolated from normal AC.23 Hypermethylation of several CpG sites of superoxide dismutase (SOD) 2 was found in OA cartilage, which correlated with decreased expression of the SOD2 gene.25

The histone modification also determines the chromatin status and thus the accessibility of genes to transcription factors. Among posttranslational modifications of nucleosomal histones, acetylation and methylation are the most studied and best characterized modifications.27, 28 Whereas acetylation generally activates transcription, lysine methylation can either activate or repress gene expression conditional to the specific modified residue.29, 30 Methylation of the histone H3 lysine 4 (H3K4) usually activates gene transcription, the di- and trimethylated forms of H3K4 being the most positively correlated. On the other hand, methylation of histone H3 lysine 9 and 27 (H3K9, H3K27) represses gene transcription.29, 30 Although the change in histone methylation patterns has been reported in the developmental process or in oncogenesis,19, 31, 32 there is a paucity of data on the change in the pathogenesis of OA. The induction of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) by IL-1 was associated with H3K4 di- and trimethylation at the iNOS and COX-2 promoters.33

The main extracellular matrix components of the articular cartilage are collagens (types II, IX, and XI) and proteoglycans (mainly aggrecan). The expressions of these genes are controlled by SOX-5, SOX-6, and SOX-9, members of a family of regulatory molecules related to the sex-determining factor named sex-determining region Y (SRY).34 These molecules maintain the chondrocytic phenotypes,34, 35 and are vital for chondrogenesis in the embryonic development.34, 36 SOX-9 is expressed in all chondroprogenitors except in hypertrophic chondrocytes.35

In this study, we focused on the epigenetic changes of SOX-9, the key chondrogenic transcription factor, and an anabolic factor, in the pathogenesis of OA. So the purpose of this study was to test the hypothesis that OA cartilage was associated with gene-inactivating epigenetic change in SOX-9 gene promoters. Methylation status was investigated in normal and OA cartilages via methylation-specific PCR (MSP) and bisulfite sequencing analysis. The effect of the demethylation on the SOX-9 gene and protein expression was also investigated by 5-azacytidine (5-AzaC) treatment. The effect of SOX-9 promoter methylation on the binding affinity of transcription factors was assessed using electrophoretic mobility shift assay (EMSA) and protein-DNA binding assay. Histone modification at SOX-9 promoter was also investigated by chromatin immunoprecipitation (ChIP).

Materials and Methods

Procurement of sample and cell culture

AC was obtained from the femoral heads of patients who had a fracture of neck of the femur (#NOF) without OA (age range 64–83 years, n = 9, four males and five females), and from those of OA patients (age range 58–69 years, n = 9, all females) undergoing total hip arthroplasty. The study was approved by the Institutional Review Board of the authors' institution. Informed consent was obtained from all donors. The femoral heads underwent gross examination and a small segment was resected for histology.37 Mankin's histologic/histochemical grading system (HHGS) was used for histological grading. Chondrocytes were isolated from AC samples as described, and cultured to passage 1.38 Briefly, the cartilage was peeled from the surface of femoral head using a scalpel, washed three times with phosphate-buffered saline (PBS) and cut into small pieces (2–3 mm3) in serum-free Dulbecco's modified Eagle's medium/Ham F-12 (DMEM/F-12; Gibco BRL, Grand Island, NY, USA). Cartilage slices were treated with 0.25% trypsin containing 1 mM EDTA (Gibco BRL) at 37°C for 15 minutes; then digested with 2 mg/mL of collagenase, 5% fetal bovine serum (FBS; Gibco BRL), 1% antibiotics (penicillin, 100 U/mL; streptomycin, 0.1 mg/mL; and amphotericin B, 0.25 mg/mL; Gibco BRL) overnight at 37°C on a gyrating shaker. The suspension was filtered through sterile nylon mesh to remove any undigested material and centrifuged at 450 g for 5 minutes. The supernatant was discarded, and the cell pellet was washed twice with PBS. Finally, the cells were resuspended in DMEM/F-12 supplemented with 10% FBS and 1% antibiotics. Cell counts were performed on a hemocytometer. Chondrocytes were seeded in 10-cm2 Falcon flasks (2 × 105 cells/cm2). Chondrocytes were cultured in DMEM/F-12 supplemented with 10% FBS and 1% antibiotics, and cultures were maintained at 37°C in a 5% humidified atmosphere.

MSP

Genomic DNA was extracted from the cartilage using a DNeasy tissue system (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Bisulfite-modified genomic DNA was also prepared using an EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA), according to the manufacturer's instructions. The PCR reaction was carried out in a volume of 20 µL with converted genomic DNA using AccuPower HotStart PCR PreMix (Bioneer, Daejeon, Korea) with 10-pmol primers (Supplemental Table 1). CpG-rich regions within the upstream sequences 5 kb from the transcription start site (TSS) were analyzed. Putative CpG-rich islands and respective primers for MSP were derived from the CpG Island Searcher (http://cpgislands.usc.edu) and the MethPrimer program (http://www.urogene.org/methprimer). The PCR reaction was carried out in a volume of 20 µL with converted genomic DNA using AccuPower HotStart PCR PreMix (Bioneer). The amplification was carried out as follows: 94°C for 5 minutes; 45 cycles at 94°C for 30 seconds; 56°C to 60°C for 30 seconds; 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. The PCR reaction was confirmed by electrophoresis in a 1.8% agarose gel, visualized by SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA) staining and the images were taken using LAS3000 (Fuji Film, Tokyo, Japan). Intensity of each PCR band was analyzed using the Image J program (National Institutes of Health, Bethesda, MD, USA).

Bisulfite sequencing analysis

Genomic DNA used in MSP was also used for bisulfite sequencing (BSQ). Primers were designed based on MethPrimer program (Table 1). PCR using bisulfite-treated genomic DNA was performed as follows; 94°C for 5 minutes; 45 cycles at 94°C for 30 seconds; 46°C for 30 seconds; 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. The PCR reaction was confirmed by electrophoresis in a 1.8% agarose gel, visualized by SYBR Safe DNA gel stain (Invitrogen) and specific bands were eluted using GeneAll Expin Gel SV kit (GeneAll, Seoul, Korea). Elute of specific PCR product was ligated with TA vector using TOP cloner TA kit (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions. After transformation of ligated products into DH5α chemically competent E. coli, clones were screened by PCR. Positive clones (n = 5–13) were cultured and plasmid DNA was isolated using the GeneAllR Exprep Plasmid SV kit according to its manual. Sequences of each plasmid were analyzed using M13R or M13F primer (Macrogen, Seoul, Korea). Sequences were analyzed using 2BLAST (http://blast.ncbi.nlm.nih.gov).

Table 1. Primer Sequences for Bisulfite Sequencing
SOX-9 promoter regionSequence (5′ to 3′) (Accession#: NG_012490)
R1F-TGTATAGATTTAAATTGAAAGGTGAT
R-CATTTTTAACTAAAAATAATTTTACCTTAA
R2F-AGGTTATTAGGGTAGATTGGAGGTT
R-ATACCACTCAAAACTATAAAATCCC
R3F-TTTTTTAGTTGTTGTTGTTTAGTTTAG
R-AATTACCCCTTATCTATTCCAATTC
R4F-TTTTATTTTGAGTATTGAGTGTTT
R-AAATCTAAAATCTCCCCCTTTC
R5F-AGGGTTTTTTTGGGGAGTTATT
R-AAAACCAAAATCCTCCATCTAATA
Proximal
 MethylatedF-GTATTAAGAAGCGGTTGTTTTTC
R-TTTCCGAAATTAAAACTAACGAA
 UnmethylatedF-GTATTAAGAAGTGGTTGTTTTTT
R-TTTCCAAAATTAAAACTAACAAA

5-AzaC treatment

Chondrocytes were plated at 2 × 105 cells/cm2 and treated 24 hours later (day 0) with 0.006% DMSO or 10 µM 5-AzaC (Sigma, St. Louis, MO, USA) in 0.006% DMSO. Media containing DMSO or DMSO + 5-AzaC was exchanged daily and lasted for 8 days. Cells were then harvested for analysis.

Tiling chip array

The chip was designed to cover anabolic genes (SOX-9, COL2A1, IGF1), covering promoter regions (∼8 kb) and inner variable region of the SOX-9 (Accession No.: NG_012490), COL2A1 (Accession No.: NG_008072), IGF-1 (Accession No.: NG_011713) genes. The information on used microarray chips is summarized in Supplemental Table 2. To enrich for methylated DNA sequences, methylated DNA immunoprecipitation (MeDIP) was conducted according to the manufacturer's process using MethylCollector Ultra (Active Motif, Carlsbad, CA, USA). The genomic DNA (gDNA) from normal and OA cartilage was differentially labeled using fluorescent dyes (Cy3/Cy5) using the Agilent Genomic DNA Enzymatic Labeling kit (Agilent Technologies, Santa Clara, CA, USA) and hybridized to the tiling arrays. The ChIP DNA was purified by PCR Purification Kit (Qiagen) for amplification. Purified immunoprecipitated DNAs were amplified using a GenomePlex Complete Whole Genome Amplification (WGA) kit (Sigma, St. Louis, MO, USA), as described by the manufacturer, differentially labeled using fluorescent dyes (Cy3/Cy5) with Agilent Genomic DNA Enzymatic Labeling kit (Agilent Technologies), and hybridized to the tiling arrays. Scans and images were analyzed using Agilent's DNA microarray scanner and Feature Extraction software. Tiling array data was analyzed with GeneSpring software (Agilent Technologies).

Real-time reverse-transcription PCR

Total RNA was isolated using the standard guanidine isothiocyanate Tri-Reagent (Sigma) protocol and quantified using the Quant-iT RNA assay kit and the Qubit Fluorometer system (Invitrogen). Using the ReverAid (Fermentas Inc., Hanover, MD, USA) H Minus first-strand synthesis for RT-PCR, 1 µg total RNA was reverse-transcribed with 0.5 µg oligonucleotide (dT) primer. All the PCR reactions were performed in standard 10-µL reaction: 4.5 µL (10 ng) cDNA, 0.5 µL 100-µM sense and 0.5 µL 100-µM antisense primers, and 4.5 µL LightCycler 480 SYBR Green I Master mix (Roche Diagnostics, Penzberg, Germany). GAPDH was used as an internal control for PCR amplification and the relative normalization ratio of PCR products derived from each target gene was calculated using software of the LightCycler System (Roche, Indianapolis, IN, USA). All experiments were performed in triplicate.

The primer pairs used are as follows:

  • SOX-9 forward (Accession No.: NM_000346): 5′-GGAGCTCGAAACTGACTGGAA-3′

  • SOX-9 reverse (Accession No.: NM_000346): 5′-GAGGCGAATTGGAGAGGAGGA-3′

  • GAPDH forward (Accession No.: NM_002046): 5′-CACATGGCCTCCAAGGAGTAA-3′

  • GAPDH reverse (Accession No.: NM_002046): 5′-GTACATGACAAGGTGCGGCTC-3′

Western blot analysis

To confirm the translation of SOX-9 gene expression at the protein level, protein extracts were analyzed by Western blotting for SOX-9. Briefly, cells were washed twice with cold PBS, and suspended in radioimmunoprecipitation assay (RIPA) lysis solution according to the manufacturer's instructions (Pierce Biotech, Rockford, IL, USA). The protein concentration was determined using a Qubit assay kit (Invitrogen) and an equal amount of protein extract was fractionated by 10% (vol/vol) SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with TBS-T (10 mM Tris; 150 mM NaCl, and 0.05% Tween 20) containing 3% (wt/vol) nonfat powdered milk, the blots were incubated with specific primary antibody for 2 hours at room temperature, washed, and incubated with secondary antibody for 1 hour at room temperature, then washed again. The blots were developed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotech) following the kit protocols. After three washes, the protein bands were visualized with an enhanced chemiluminescence (ECL) Western blot analysis system (Amersham Biosciences, Piscataway, NJ, USA). The primary antibody was rabbit SOX-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:2000 in TBST/2.5% nonfat dry milk. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-rabbit diluted 1:2000 in TBS-T 2.5% powdered nonfat dry milk. The intensity of bands were quantified using an image processing program (ImageJ, NIH, Bethesda, MD, USA) and the relative density of SOX-9 bands to β-actin bands was determined. Also, to confirm modified histone, soluble chromatins were analyzed for H3 tri-methyl K9, H3 tri-methyl K27, and H3 acetyl (K9 + K14 + K18 + K23 + K27). The nuclear extracts were also analyzed for transcription factors cAMP response element-binding (CREB) and CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) to augment the result of EMSA at the protein level. The primary antibodies were rabbit anti-H3 tri-methyl K9 polyclonal antibody (Abcam, Cambridge, UK), mouse anti-H3 tri-methyl K27 monoclonal antibody (Abcam), rabbit anti-H3 acetyl (K9 + K14 + K18 + K23 + K27) polyclonal antibody (Abcam), rabbit anti-nuclear transcription factor Y subunit beta (NFYB) polyclonal antibody (Acris Antibodies, San Diego, CA, USA), and rabbit anti-CREB polyclonal antibody (Abcam) diluted, respectively, 1:2000 in TBST/2.5% nonfat dry milk. The secondary antibody was HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) (Abcam) or goat anti-mouse IgG (Abcam) diluted 1:2000 in TBS-T 2.5% powdered nonfat dry milk. This experiment was repeated in three samples, each from different individuals.

ChIP and indirect assessment of histone modification by PCR

Chromatin preparation in normal and OA primary chondrocytes was carried out using ChromaFlash Chromatin Extraction Kit (Epigentek, Farmingdale, NY, USA) as described by the manufacturer. The soluble chromatins were sonicated 15 minutes on ice 3× and incubated with 30 µL protein A/G Sepharose (50% slurry; GE Healthcare Bio-Sciences Corp., Uppsala, Sweden) under gentle agitation 2 hours at 4°C. The supernatant was transferred to a new microcentrifuge tube, followed by immunoprecipitation with 1 µg of anti-H3 tri-methyl K9, anti-H3 tri-methyl K27, anti-H3 acetyl (K9 + K14 + K18 + K23 + K27) antibody, and non-immune rabbit IgG (Santa Cruz Biotechnology) as control at 4°C overnight. Protein A/G Sepharose (30 µL of a 50% slurry) was then added and incubated for 1.5 to 2 hours. The pellets were successively washed with different buffers (low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, TE buffer). Protein-DNA cross-links were reversed by overnight incubation at 65°C in 100 µL elution buffer (1% SDS in TE). DNA was purified using a PCR purification kit (Qiagen) and eluted in 50 µL of elution buffer. The immunoprecipitated DNA was amplified by real-time PCR using SYBR Green Ex-Taq Master Mix (Takara, Shiga, Japan) and PCR using AccuPower HotStart PCR PreMix (Bioneer). Primers were designed for the SOX-9 proximal promoter. Also, the soluble chromatins were used for other analyses (Western blot analysis, quantification of histone H3 modification).

The primer pairs used are as follows:

  • SOX-9 proximal promoter forward (−307 to −287): 5′-GCGGAGAAGGCACTAAAATTC-3′

  • SOX-9 proximal promoter reverse (−201 to −185): 5′- CAAAGGGCGGACGGTAG-3′

EMSA

In vitro DNA binding activity for transcription factors (CREB and CBF/NF-Y) were assayed by incubating about 6 µg of normal and OA primary chondrocytes nuclear extracts. Synthetic oligonucleotides were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer's instructions and annealed for 30 minutes at room temperature. Binding reactions were carried out for 20 minutes at room temperature in the presence of 50 ng/µL poly(dI-dC), 0.05% Nonidet P-40, 5 mM MgCl2, 10 mM EDTA, and 2.5% glycerol in 1× binding buffer (LightShift Chemiluminescent EMSA kit; Pierce) using 20 fmol of biotin-end-labeled target DNA and 6 µg of nuclear extract. Two microliters (2 µL) of anti-CREB (Abcam) or anti-NFYB antibody (Acris Antibodies) was added per 20 µL of binding reaction where indicated. Assays were loaded onto native 10% polyacrylamide gels pre-electrophoresed for 30 minutes in 0.5× TBE and electrophoresed at 100 V before being transferred onto a positively charged nylon membrane (Hybond-N+; Amersham) in 0.5× TBE at 100 V for 45 minutes. Transferred DNAs were cross-linked to the membrane at the transilluminator and detected using HRP-conjugated streptavidin (LightShift Chemiluminescent EMSA kit) according to the manufacturer's instructions.

The transcription factor binding site probe pairs were as follows:

  • Unmethylated CRE 1/2-site forward: 5′-CCCGCCTCGTCACCCAGCCC-3

  • Unmethylated CRE 1/2-site reverse: 5′-GGGCTGGGTGACGAGGCGGG-3′

  • Methylated CRE 1/2-site forward: 5′-CCCGCCTCGTCACCCAGCCC-3′

  • Methylated CRE 1/2-site reverse: 5′- GGGCTGGGTGACGAGGCGGG-3′

  • Unmethylated CCAAT box 2 forward: 5′-TGCTGTGATTGGCGGGTGG-3′

  • Unmethylated CCAAT box 2 reverse: 5′-CCACCCGCCAATCACAGCA-3′

  • Methylated CCAAT-2 box 2 forward: 5′-TGCTGTGATTGGCGGGTGG-3′

  • Methylated CCAAT-2 box 2 reverse: 5′-CCACCCGCCAATCACAGCA-3′

Protein-DNA binding assay

In vitro DNA binding activity for transcription factors (CREB, CBF/NF-Y) were also quantified using EpiQuik General Protein-DNA Binding Assay Kit (Epigentek) according to the manufacturer's instructions.

Quantification of histone H3 modification

Quantification of histone H3 modification was performed using EpiQuik Global Pan-Methyl Histone H3K9 Quantification Kit, EpiQuik Global Pan-Methyl Histone H3K27 Quantification Kit, and EpiQuik Global Histone H3 Acetylation Assay Kit (Epigentek) according to the manufacturer's instructions.

Statistical analysis

Descriptive statistics were used to determine group means and SDs. Quantitative data were statistically analyzed using t test with p values < 0.05 as being statistically significant.

Results

Gross and histological findings from AC of femoral heads

Gross examination revealed that none of the #NOF samples had sign of OA such as softening, fibrillation, and eburnation, which were observed in OA samples. The histological finding also demonstrated that AC of #NOF samples were not associated with histological features observed in OA samples. The Mankin score was 10.5 ± 2.5 in OA samples and 1.2 ± 0.8 in #NOF samples (Supplemental Fig. 1).

Screening for methylation status of anabolic genes in normal and OA cartilages via tiling chip array and MSP

We first checked the methylation status for the promoter regions covering approximately −5.0 kb from the TSS of anabolic genes (SOX-9, COL2A1, IGF-1) via MSP and MeDIP from genomic DNA isolated from normal and OA cartilage of the femoral heads. Only one to three sites were available for the MSP from the promoters of COL2A1 and IGF-1 because these promoters had only two to three CpG islands. From MSP, there was not a significant difference in the methylation status between normal and OA samples in the regions of interest (ROIs) at these promoters (Supplemental Fig. 2). The result of MeDIP also demonstrated no increase in methylation at these promoters (Supplemental Fig. 3). As SOX-9 promoter has six CpG islands up to the promoter site of −5.0 kb, eight ROIs were available for MSP (Fig. 1A). Methylation was increased in OA cartilage compared to normal in the R3 (from −3653 to −3496, p = 0.0186) and R4-1 (from −3111 to −2983, p = 0.0014) of SOX-9 promoter (Fig. 1B, C). Tiling chip array of CpG site performed to detect global methylation status showed a general increase in methylation at six CpG islands of SOX-9 promoters in OA cartilage (Fig. 1D).

Figure 1.

Methylation profiling of SOX-9 by methylation-specific PCR (MSP). (A) Scheme of CpG islands and region of interest detected by MSP. TSS = transcription start site. (B) Results of MSP on SOX-9 promoter from gDNA isolated from normal and OA cartilage (M = methylated primer; U = unmethylated primer). (C) Dot plots showing the methylation index [intensity of methylated band/(intensity of methylated band + intensity of unmethylated band)] in regions detected by MSP. R1–R7: regions for MSP. n = 9, *p < 0.05, **p < 0.01. (D) Methylation of CpG islands on SOX-9 promoters of OA cartilage as detected by tiling chip arrays. Data are shown as the percentage over the control (normal cartilage from #NOF). Bars represent mean ± SD.

Detailed investigation on the methylation status of SOX-9 promoter by bisulfite sequencing

Because MSP detects the methylation status of one specific CpG site only, we next analyzed the methylation status of individual CpG sites of five areas located from −4548 to −2846 in the SOX-9 promoter by bisulfite sequencing (BSQ1–5) because that area was found to have the highest degree of difference in comparing normal to OA, as shown in MSP (Fig. 2A). Methylated CpG sites were significantly increased in all examined regions in OA cartilage (Fig. 2B, C). In order to see the correlation of increased SOX-9 promoter methylation to SOX-9 gene and protein expression, endogenous levels of SOX-9 expression were investigated in the chondrocytes isolated from normal and OA cartilage. SOX-9 mRNA in OA chondrocytes were one-half that of normal chondrocytes (Fig. 2D). Protein expression of SOX-9, as shown by Western blot analysis, also demonstrated a decrease in OA chondrocytes (Fig. 2E).

Figure 2.

The methylation status of SOX-9 promoter by bisulfite sequencing. (A) Scheme of bisulfite sequencing (BSQ) analysis for SOX-9 promoter (BSQ1–5; regions from −4548 to −2846, TSS = transcription start site). (B) Bisulfite sequencing analysis for SOX-9 promoter from normal and OA cartilage (black circle: methylated form; open circle: unmethylated form in CpG site). (C) Percentage of methylated CpG sites from BSQ analysis. Last panel represents total methylation rates (methylated CpG sites per total analyzed clone number × total CpG sites) in normal and OA cartilage. Bars represents mean ± SD. n = 5, ***p < 0.001. (D) Results of qRT-PCR for SOX-9 mRNA in normal and OA chondrocytes. Data are shown as the percentage over the control (normal cartilage from #NOF). Bar represents mean ± SD. n = 9, ***p < 0.001. (E) Western blot analysis and relative quantification of SOX-9 to β-actin in normal and OA chondrocytes. Bar represents mean ± SD. n = 3, ***p < 0.001. β-actin was used as a housekeeping protein.

Effect of 5-AzaC on the methylation status of SOX-9 promoter

We also examined if the methylation status of SOX-9 promoter could be altered by 5-AzaC, a DNA methyltransferase inhibitor, and also if the modification could cause a change in the gene and protein expressions of SOX-9.

The treatment of 5-AzaC decreased the methylation in all of six CpG islands of SOX-9 promoter in OA cartilage in the MeDIP (Fig. 3A). The results from real-time qPCR showed that SOX-9 mRNA increased in OA chondrocytes to a level comparable to normal chondrocytes when treated 5-AzaC (Fig. 3B). Western blot also confirmed that 5-AzaC treatment increased the expression of SOX-9 protein in OA (Fig. 3C).

Figure 3.

Effect of 5-azacytidine (5-AzaC) on the methylation status of SOX-9 promoter. (A) Methylated DNA immunoprecipitation (MeDIP) in normal, untreated and 5-AzaC treated OA chondrocytes, respectively. (B) qPCR for SOX-9 mRNA in normal, untreated and 5-AzaC treated OA chondrocytes. The values were expressed as the percentage over the control (chondrocytes from normal cartilage from #NOF). Bars represent mean ± SD. n = 9, ***p < 0.001. (C) Western blot analysis and relative quantification of SOX-9 to β-actin in normal, untreated, and 5-AzaC–treated OA chondrocytes. Bar represents mean ± SD. n = 3, ***p < 0.001. β-actin was used as a housekeeping protein.

Methylation of SOX-9 promoter affecting the binding affinity of transcription factors

The sequence of the human SOX-9 proximal promoter region from −315 to +5 bp relative to the TSS was previously mapped.39 Within this region, there are several predicted transcription factor binding sites including two Sp1-like sites, a CRE 1/2-site, and two CCAAT boxes.39 In order to determine if a methylation within the SOX-9 proximal promoter region affected the binding affinity of transcription factors, we investigated whether the CCAAT box 2 and CRE 1/2-site within the SOX-9 proximal promoter specifically interacted with CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) and cAMP response element-binding (CREB) in nuclear extracts from normal and OA chondrocytes (Fig. 4A). From bisulfite sequencing of SOX-9 proximal promoter using primers listed in Table 1, increased methylation had been confirmed on CCAAT box 2 and CRE 1/2-site in OA chondrocytes versus normal (n = 3, each: Supplemental Fig. 4).

Figure 4.

Methylation of SOX-9 promoter affects the binding affinity of transcription factors. (A) Sequence of human SOX-9 proximal promoter region from −315 to +5 bp and binding sites for transcription factors. (B) EMSA for transcription factor CBF/NF-Y and CREB to CCAAT box 2 and CRE 1/2-site, respectively. Western blotting was also performed to further determine the identity of the binding factors (lower panels). M = methylated probe; U = unmethylated probe. (C) Binding affinity of CBF/NF-Y to CCAAT box 2, and CREB to CRE 1/2-site from ELISA and Western analysis. The values were expressed as the percentage over the control (chondrocytes from normal cartilage from #NOF). Bars represent mean ± SD. n = 3, **p < 0.01, ***p < 0.001.

EMSA for CBF/NF-Y and CREB binding was performed with a probe containing the unmethylated or methylated CCAAT box 2 and CRE 1/2-site of SOX-9 promoter. Stronger binding complexes were formed with unmethylated CCAAT box 2 and CRE 1/2-site probes than with methylated ones in both normal and OA chondrocytes. In order to further determine the identity of the factors bound to the CCAAT box 2 and CRE 1/2-site, Western blotting was also performed using anti-NFYB and anti-CREB antibodies (Fig. 4B). It also showed a greater amount of DNA probe-protein complex in the unmethylated probes compared to the methylated ones in both normal and OA chondrocytes (Fig. 4B).

Next, in order to further quantify the binding of transcription factors to SOX-9 proximal promoters in actual conditions, the DNA-protein binding affinity was measured using protein-DNA binding assay. Unmethylated CCAAT box 2 and CRE 1/2-site probes were reacted with nuclear extracts from normal chondrocytes whereas methylated CCAAT box 2 and CRE 1/2-site probes were reacted with nuclear extracts from OA chondrocyte. Unmethylated CCAAT box 2 or CRE 1/2-site probe, respectively, formed a strong complex with CBF/NF-Y or CREB from normal chondrocytes compared to the methylated probes with CBF/NF-Y or CREB from OA chondrocytes. Western blot analysis also confirmed a greater amount of DNA probe-protein complex in the normal chondrocyte-unmethylated probe coupling than in the OA chondrocyte-methylated probe (Fig. 4C).

Differences in histone modification of SOX-9 promoter

As the last step in the evaluation of epigenetic change, the changes in the histone modification including methylation and acetylation were investigated. First, by ELISA and Western blotting, we assessed the global methylation in the 9th and 27th lysine residue of histone 3 (H3K9, H3K27), which are known to inactivate gene transcription. There were small, but significant increases of both H3K9 and H3K27 in OA chondrocytes compared to normal chondrocytes (Fig. 5A). The methylation of H3K9 and H3K27 at SOX-9 promoters was investigated using tiling chip array, and PCR from ChIP fragments. There were significant increases in H3K9 and H3K27 trimethylation on SOX-9 promoter areas (Fig. 5B). Acetylations at the 9th, 15th, 18th, 23rd, and 27th lysine residue of histone 3, which activates gene transcription, were also assessed. The results from ELISA and Western blot analysis showed a significant decrease in global acetylation in OA chondrocytes compared to normal chondrocytes (Fig. 5C). The acetylation of H3K9, 15, 18, 23, and 27 at SOX-9 promoters were also significantly decreased in OA chondrocytes (Fig. 5D)

Figure 5.

Analysis of histone modification in normal and OA chondrocytes. (A) Global methylation of H3K9 and H3K27 by ELISA and Western blot analysis. (B) The methylation rate of H3K9 and H3K27 at SOX-9 promoters investigated using tiling chip array (left panel) and qPCR from ChIP fragments (center and right panels). (C) Global acetylations at the 9th, 15th, 18th, 23rd, and 27th lysine residue of histone 3 by ELISA and Western blot analysis. (D) The acetylation of H3K9, 15, 18, 23 and 27 at SOX-9 promoters investigated using tiling chip array (left panel) and qPCR from ChIP fragments (right panel). In all panels, values are shown as the percentage over the control (normal chondrocytes). Bars represent mean ± SD. n = 3, **p < 0.01, ***p < 0.001.

Discussion

In this study, we tested the hypothesis that OA was associated with a change in the epigenetic status of SOX-9. The overall results showed increased methylation of SOX-9 gene promoter as well as increased methylation of gene-inactivating histone residues (H3K9 and H3K27) and decreased histone acetylation. Although it is not clear whether these changes are the cause or the results of the disease, the change of epigenome can explain the decreased SOX-9 gene and protein in OA, which are shown here and also reported in previous studies.38, 40, 41

The present study starts from the typical theory in the pathogenesis of OA that the expression of anabolic genes is decreased whereas that of catabolic genes is increased.5, 6 Considering that decreased methylation of catabolic gene promoters had been already reported,7 we were more interested on the epigenetic status of anabolic genes. Therefore, we screened several anabolic genes including IGF-1 and COL2A1 in addition to SOX-9 in normal and OA cartilages. Since significant differences were not found in other genes via MSP analysis and tiling array, we focused on SOX-9, with a growth of evidence showing that SOX-9 was the key transcription factor for chondrogenesis,34, 35 and that OA was associated with downregulation of SOX-9 expression.38 Our results imply that the increased methylation of SOX-9 promoter, the gene-inactivating changes in histone modification, and the subsequent decrease in SOX-9 gene and protein expression are involved in the pathogenesis of OA. However, the cause-effect relationship between the epigenetic change in SOX-9 promoters and the OA progression is left to a further study.

Whereas OA most frequently develops in the knee joint, the hip joint was chosen in this study due to the difficulty of obtaining normal cartilage from the knee joint. Because of this difficulty, our previous study used minimally osteoarthritic cartilage from the knee joint of the same patient as the control and found no difference in the methylation between the minimal and advanced OA.38 The availability of cartilage from the femoral head obtained from patients who underwent arthroplasty from a fracture of neck of the femur enabled the design of present study. Although #NOF patients were significantly older than OA patients, the gross and histological findings of their femoral head guaranteed that the AC ranged within the normal.

It can be claimed that the AC from #NOF is not a suitable control for OA cartilage. Da Silva and colleagues22 found that chondrocytes of the superficial zone expressed proteases such as OA chondrocytes, whereas those from intermediate and deep zones in the AC of #NOF did not. They thus insisted that only intermediate and deep zones constituted suitable control cartilage for a study on protease expression.22 We were not able to collect respective samples from different AC layers because it was practically difficult to distinguish one layer of AC from another in the process of obtaining an AC sample from the femoral head. It can be speculated that the superficial layer of #NOF samples had epigenetic profile similar to OA cartilage. Nevertheless, consistent results from a relatively large number of patients in the present study unambiguously indicated a definite difference in the epigenetic status between normal and OA cartilages in overall.

Another strength of this study lies in the extensive search of the promoter site (∼5 kb) of SOX-9 promoter. A previous study investigated SOX-9 promoter during chondrogenic differentiation of synovium-derived cells and reported low methylation state. However, they analyzed CpG-rich region in ∼1 kb upstream sequence of SOX-9 promoter.42 Our bisulfite sequencing analysis of broader SOX-9 promoter region indicated that CpG sites from −4548 to −2846 were highly methylated in OA cartilages compared to normal cartilage from #NOF.

The treatment of 5-AzaC, which is a potent inhibitor of DNA methyltransferase 3a, provides a unique opportunity to watch whether the inhibition of transcription associated with increased methylation of the promoter site can be actually reversed and thus determine if increased methylation causes the inhibition of transcription in the specific gene. Our results definitely showed that the low level of SOX-9 mRNA in OA chondrocytes could be reversed through inhibition of DNA methylation. In addition, the decreased binding affinity of methylated SOX-9 proximal promoter to transcription factors such as CBF/NF-Y and CREB explains the low expression of SOX-9 gene and protein in advanced OA as shown in the present and previous studies.38, 40, 41

The methylation at lysine residues of histone has been known to induce either euchromatin or heterochromatin status depending on the residue. Although the status of histone methylation has been widely investigated in the development of cancer,43–45 it has not been broadly reported in relation to the pathogenesis of OA. Our results clearly demonstrated the difference in histone methylation and acetylation between normal and OA cartilage. The increased methylation of H3K9 and H3K27 and decreased acetylation in SOX-9 promoters also attest to the decreased SOX-9 gene expression in OA cartilage.

In conclusion, we found that hip OA was associated with a change in the epigenetic status of SOX-9 promoter including increased DNA methylation, increased methylation of gene-inactivating histone residues (H3K9 and H3K27), and decreased histone acetylation. This finding may present another clue in unraveling the pathogenesis of OA.

Disclosures

All authors state that they have no conflicts of interest.

Acknowledgements

This work was supported by a grant from the Ministry of Health and Welfare in Korea (A100010). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' roles: Study design: KIK and GII. Data collection: KIK. Data analysis: KIK and GII. Data interpretation: KIK, YSP, and GII. Drafting manuscript: KIK and GII. Revising manuscript content: KIK and GII. Approving final version of manuscript: KIK, YSP, and GII.

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