Osteoarthritis (OA) is an age-associated multifactorial disease characterized by joint dysfunction and cartilage degeneration that is widely prevalent in the elderly population (). 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 (). 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 associated with gene silencing (). 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 pathways (). 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 neurons ([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) (). All of these intermediates 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 apolipoprotein B messenger RNA–editing enzyme catalytic polypeptide–like (APOBEC) family of DNA deaminases that can act independently on 5mC or deaminate 5hmC after TET action, leading ultimately to DNA demethylation ().
A critical role of 5hmC and TET proteins has emerged in stem cell differentiation and embryonic development ([13, 14]) as well as in cancer. Mutations in TET1 and TET2 were initially associated with various forms of leukemia ([15, 16]). Intriguingly, a global loss of 5hmC was observed in multiple cancers, including hematologic disorders and solid tumors, such as colon, prostate, and breast cancers. In melanoma, the loss of 5hmC is a direct result of the loss of TET function (). Gain of TET2 activity was shown to not only restore the 5hmC epigenome, but also suppress the tumor, demonstrating that 5hmC homeostasis is dynamic and its perturbation is a causal factor in melanoma ().
Evidence has been accumulating with regard to a role of DNA methylation in the pathogenesis of OA ([3, 18]). Alterations in DNA methylation patterns have been observed in OA chondrocytes, particularly a loss of DNA methylation of the promoters of various OA-associated genes, including matrix metalloproteinase 3 (MMP-3), MMP-9, MMP-13, ADAMTS-4, and interleukin-1β (IL-1β) ([19-22]). Expression of growth differentiation factor 5, the OA susceptibility gene, is modulated by DNA methylation (). In addition, a recent study has reported DNA demethylation at cytokine-responsive enhancer elements of inducible nitric oxide synthase to be critical for gene induction (). In view of the recent advances in the dynamics of DNA methylation, we sought to investigate whether 5hmC and the active DNA demethylation machinery play a role in the pathogenesis of OA.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
OA has a complex pathogenesis, affected by both genetic and environmental factors, and the early causal and sequential events during OA development are still unclear (). Our understanding of the epigenetic changes that take place during the initiation and progression of OA is also limited. Previous work has suggested a role of DNA demethylation in “unsilencing” OA-associated enzymes ([3, 18]). Our study provides the first evidence of a dysregulation of the DNA methylation dynamics in OA. It is surprising that a significant down-regulation of TET1 is concomitant to an increase in 5hmC in OA chondrocytes, considering that a loss of TET1 in melanoma, prostate, and breast cancer leads to an opposite effect, a global loss of 5hmC ([17, 36]). These observations, however, underscore the dynamic nature of the global 5hmC homeostasis and its profound effect on transcription and cellular fate in pathologic conditions.
All of the TET family members, TETs 1, 2, and 3, are capable of the conversion of 5mC to 5hmC, as well as further oxidation to 5fC and 5caC intermediates (). TET1 function, however, appears to be distinct from that of TETs 2 and 3 in OA pathogenesis, since only TET1 expression was down-regulated in OA chondrocytes. However, TET function involves multiple known and hitherto unknown cofactors, and hence, it remains possible that the function of TET2 or TET3 is perturbed in OA chondrocytes independently of their gene expression. A decrease in TET1 function can potentially lead to 5hmC accumulation if TET1 is the major contributor to the conversion of 5hmC to 5fC or 5caC; however, the exact role of the TET proteins in the stepwise generation of 5hmC as well as its removal (by conversion to 5fC and 5caC) is still undetermined.
MMPs play important roles in diverse biologic processes and pathologies, including cancer and arthritis (both OA and RA), and their transcriptional regulation has thus been widely studied (). The global increase in 5hmC translates to locus-specific 5hmC enrichment in both MMP-1 and MMP-3, which play significant roles in OA pathology. The promoters of most MMPs, including MMPs 3, 9, and 13, do not contain dense CpG regions. Although initially, differential methylation of CpG islands was mainly thought to influence gene expression, it is now known that low-density CpG regions contribute extensively to tissue-specific gene regulation ([40, 41]). In addition, while 5mC enrichment appeared to be regulatory mostly in promoters and distal regions, the presence of 5hmC in gene bodies shows a strong positive correlation with high levels of gene expression in embryonic stem cells and neurons ([31, 42-45]).
Our studies demonstrate that 5hmC enrichment at specific CpG sites in the MMP-1 and MMP-3 promoters correlates with increased gene expression in OA chondrocytes. These observations are consistent with studies showing that 5hmC enrichment at promoters and gene bodies correlate with high levels of gene expression (). A study in OA patients had previously demonstrated significantly higher DNA demethylation associated with the –36-bp site for MMP-9, the –635-bp site for MMP-3, and the –110-bp site for MMP-13 promoters in OA patients (). However, the restriction enzymes used in this study did not distinguish between 5mC and 5hmC; therefore, the changes in 5mC were likely to have been understated.
The MMP-3 site used in our study is the Hpa II site (similar to the –686-bp site in the previous study; now designated –541 bp based on updated sequence) that is not demethylated in OA chondrocytes. Significant hydroxymethylation at this site was observed in 20–40% of OA chondrocytes, with a concomitant decrease in methylation. Consistent with a previous report (), we did not observe any increase in unmethylated cytosines. This site is proximal to the CpG site undergoing significant DNA demethylation in OA chondrocytes (–635 bp), suggesting a role of 5hmC in active DNA demethylation. Both the Hpa II sites we studied in the MMP-1 promoter showed an increase in hydroxymethylation with a decrease in methylation.
A contradiction to the observations in OA chondrocytes is that inflammatory cytokines can cause an up-regulation of MMPs 1 and 3 in normal chondrocytes without any associated increase in 5hmC in their promoters. However, the exact gene expression levels of MMPs 3 and 13 were many times higher (100–1,000-fold) in the OA patients as compared to the short-term cytokine-stimulated chondrocytes (Figure 1). Therefore, it remains possible that 5hmC enrichment in OA leads to higher MMP gene expression. Another possibility is that longer-term incubation with the cytokines and interrogation of their effects on the OA chondrocytes that may respond differently from the “normal” chondrocytes used in our study, is required to recapitulate the 5hmC dynamics in OA. In addition, it is important to analyze other CpG sites in the MMP genes, especially in the gene bodies and exons, to clarify the role of 5hmC in regulating MMP gene expression in OA.
One of the interesting insights of this study is that inflammatory cytokines can modulate TET1 expression. Analyses in OA chondrocytes as well as in short-term cytokine-treated chondrocytes, clearly showed that down-regulation of TET1 expression (both mRNA and protein) correlates with an up-regulation of the expression of MMPs 1, 3, and 13. These observations implicate TET1 as a novel modulator of OA pathology. Further analyses of the role of TET1 in normal and OA chondrocytes would be required to understand whether MMPs constitute direct or indirect targets of TET1 or whether TET1 and MMPs are modulated by a common upstream regulator. Regulation of TET1 expression by inflammatory cytokines could be a common nexus for epigenetic changes observed in cancer, OA, and possibly RA and other inflammation-associated disorders. The effect of aging on 5hmC levels is another area that needs further experimentation. Although there did not appear to be an age-associated increase in the 5hmC levels in the normal chondrocytes we tested, the disparity in the age range of the normal subjects and OA patients would not allow us to completely rule out an effect of aging based on the present observations. Detailed experimentation in a mouse model, as has been conducted for assessments of postnatal neurodevelopment and aging (), will be useful for addressing how normal aging affects 5hmC regulators and homeostasis in cartilage.
Enrichment in 5hmC in a few genes associated with OA suggests that it is a potential regulator of altered gene expression in OA; however, a broader understanding of its role will be provided by in-depth genome-wide analyses of the distribution of 5hmC and the subsequent effects on gene expression in normal and OA chondrocytes. Antibody-based approaches, using DNA enrichment with specific anti-5hmC and anti-5mC antibodies followed by high-throughput sequencing, have already been used in embryonic stem cells and neurons to map the distribution of 5hmC ([31, 42, 44-46]). Enrichment of 5hmC in euchromatic regions, at promoters with intermediate (not high) CpG density and in gene bodies of actively transcribed genes, are the common features observed in these studies. The precise effects of the location of 5hmC on transcriptional regulation and its modulation by other chromatin modifications in a context- and tissue-dependent manner are areas of vigorous investigation. Given the intriguing association of 5hmC with fundamental processes such as stem cell differentiation and pathologies such as cancer and OA, future studies will provide fundamental insights into the biology of 5hmC as well as suggest approaches for therapeutic interventions.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bhutani had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Taylor, Smeriglio, Bhutani.
Acquisition of data. Taylor, Smeriglio, Dhulipala, Rath.
Analysis and interpretation of data. Taylor, Smeriglio, Bhutani.