A global increase in 5-hydroxymethylcytosine levels marks osteoarthritic chondrocytes.
OBJECTIVE
To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA).


METHODS
Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation.


RESULTS
A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment.


CONCLUSIONS
This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.
Stanford University, School of Medicine, Stanford, California.
Sarah E B
SE
Taylor
Piera
P
Smeriglio
Lakshmi
L
Dhulipala
Madhusikta
M
Rath
Nidhi
N
Bhutani
D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D051722:ADAM Proteins / Q000235:genetics; D000071116:ADAMTS5 Protein; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D059549:Anterior Cruciate Ligament Reconstruction; D019645:Arthroplasty, Replacement, Knee; D015415:Biomarkers / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D019902:Chondrocytes / Q000201:enzymology / Q000378:metabolism*; D018899:CpG Islands; D016207:Cytokines; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007223:Infant; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D008875:Middle Aged; D006899:Mixed Function Oxygenases; D020370:Osteoarthritis, Knee / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D055815:Young Adult
D015415:Biomarkers; D016207:Cytokines; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D007041:Hypoxanthine Phosphoribosyltransferase; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1