ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 1, January 2014, pp 90–100 DOI 10.1002/art.38200 © 2014, American College of Rheumatology A Global Increase in 5-Hydroxymethylcytosine Levels Marks Osteoarthritic Chondrocytes Sarah E. B. Taylor, Piera Smeriglio, Lakshmi Dhulipala, Madhusikta Rath, and Nidhi Bhutani 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 pa- tients undergoing anterior cruciate ligament reconstruc- tion. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene ex- pression changes were monitored by quantitative poly- merase 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 glucosyla- tion and enzyme digestion–based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were moni- tored in normal chondrocytes stimulated with inflam- matory cytokines to identify the effect of joint inflam- mation. Results. A global 5–6-fold increase in 5hmC con- comitant 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 in- flammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrich- ment. Conclusion. This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accu- mulation. Our experiments identify 5hmC and its reg- ulators as potential diagnostic and therapeutic targets in OA. Osteoarthritis (OA) is an age-associated multi- factorial disease characterized by joint dysfunction and cartilage degeneration that is widely prevalent in the elderly population (1). Clinical management of this disorder is largely limited to pain management or an eventual total joint replacement. Various genes render susceptibility to OA; however, there is not a single consensus genetic basis for the disease (2). Insight into the early epigenetic changes leading to the altered gene expression in OA can provide a novel target axis for OA pathology (3,4). DNA methylation is a key epigenetic mark asso- ciated with gene silencing (5). Studies conducted during the last few years have brought about a paradigm shift in our understanding, elucidating the fact that active DNA demethylation is more dynamic and prevalent than was previously appreciated and involves DNA repair path- ways (6). DNA hydroxymethylation of the cytosine base (5hmC; currently referred to as the “sixth base”), has been discovered to be stably present in most tissues and particularly abundant in embryonic stem cells and neu- rons (6,7). The TET family of proteins, consisting of TETs 1, 2, and 3, converts 5mC to 5hmC (7,8) and can also further oxidize 5hmC to 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) (9). All of these intermedi- ates are substrates for thymine DNA glycosylase (TDG), leading to replacement by an unmodified cytosine and resulting in active DNA demethylation (10,11). Another possible route for active DNA demethylation involves the activation-induced deaminase (AID) or apolipopro- tein B messenger RNA–editing enzyme catalytic polypeptide–like (APOBEC) family of DNA deami- nases that can act independently on 5mC or deaminate Sarah E. B. Taylor, PhD, Piera Smeriglio, PhD, Lakshmi Dhulipala, BS, Madhusikta Rath, MS, Nidhi Bhutani, PhD: Stanford University, School of Medicine, Stanford, California. Address correspondence to Nidhi Bhutani, PhD, Stanford University, School of Medicine, Department of Orthopaedic Surgery, 300 Pasteur Drive, Edwards Building, Room 164, Stanford, CA 94305-5341. E-mail: nbhutani@stanford.edu. Submitted for publication May 17, 2013; accepted in revised form September 12, 2013. 90