5-hmC Leading the Pain in Osteoarthritis
Over 27 million Americans are estimated to be living with osteoarthritis (OA), a debilitating joint disorder that results from continual loss of cartilage that cushions the bones. OA is accompanied by increased levels of the cytokines Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in cartilage cells called chondrocytes. These cytokines have been shown to induce the expression of genes involved in cartilage degradation, and the promoter regions of those genes have been found to be hypomethylated in OA chondrocytes when compared to normal chondrocytes, suggesting epigenetics likely plays a role in OA progression. Active DNA demethylation is mediated by the ten-eleven translocation (TET) enzymes, which oxidize 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), and ultimately, the modification is removed. Interestingly, a recent study by Haseeb et al. found that cultured human primary chondrocytes treated with IL-1β and TNF-α had reduced global 5-hmC levels compared to untreated chondrocytes. Therefore, the authors sought to determine if these reduced 5-hmC levels were a result of IL-1β and TNF-α modulation of the TET1 oxidation pathway and if loss of 5-hmC was seen at specific regions of the genome.
Using real-time PCR to measure TET1 mRNA levels, the authors found that decreased 5-hmC levels were accompanied by a 10-fold decrease in TET1 gene expression after IL-1β and TNF-α treatment of chondrocytes. In addition, TET enzyme activity was found to be reduced by 35% in the cells treated with both cytokines, suggesting that lower 5-hmC levels can be attributed to reduced abundance and activity of TET enzymes. Furthermore, the authors showed that the effects of IL-1β and TNF-α require activation of the NF-κB pathway because chondrocytes treated with both cytokines and NF-κB inhibitors did not result in either reduced global 5-hmC levels or decreased TET1 gene expression. This suggested that activation of NF-κB negatively regulates TET1 expression. Efficient TET oxidation of 5-mC to 5-hmC also requires the co-factor α-ketoglutarate (α-KG), but in the presence of IL-1β and TNF-α, α-KG level was 40% lower compared to untreated chondrocytes as measured by LC-MS/MS. The isocitrate dehydrogenases (IDHs) enzymes, which synthesize α-KG, were also found to have lower expression and activity levels. These results demonstrate how IL-1β and TNF-α modulate 5-hmC levels by reducing TET1 and IDH activity.
Lastly, the authors were able to compare 5-hmC levels at specific gene loci between IL-1β-treated and untreated chondrocytes at single-nucleotide resolution by using a genome-wide DNA hydroxymethylation analysis called reduced representation hydroxymethylation profiling (RRHP). RRHP is a positive-display method that detects 5-hmC at CCGG sites across the genome, and 5-hmC levels can be compared between samples based on the number of sequencing reads generated at a specific CCGG site. Overall, IL-1β treated cells have a lower number of 5-hmC reads compared to untreated cells at specific loci, indicating a decrease in DNA hydroxymethylation. In comparison to untreated chondrocytes, treated cells had fewer reads at the promoter regions of five selected IL-1β-induced genes, SOX9, NOS2, ADAMTS4, COX2, and IL-6, indicating reduced 5-hmC levels at these loci. Interestingly, the relative expression of those genes increased in the presence of the cytokines, indicating that the reduction of 5-hmC levels correlated with increased gene expression.
In summary, the authors reported that the cytokines IL-1β and TNF-α reduce 5-hmC levels by interfering with TET1 and IDH enzyme expression and activity. This is correlated with reduced hydroxymethylation at specific promoter regions and, therefore, increased expression of genes known to be induced by Il-1β and TNF-α. These results demonstrate how pro-inflammatory cytokines can induce epigenetic modifications to regulate expression of genes that contribute to OA progression, and also shed light on how the 5-hmC epigenetic modification contributes to regulation of gene expression.
To learn more about the RRHP method used in this study, click here.
Haseeb A, Makki MS, & Haqqi TM (2014). Modulation of Ten Eleven Translocation 1 (TET1), Isocitrate Dehydrogenases (IDHs) expression, α-ketoglutarate (α-KG) and DNA hydroxymethylation levels by IL-1β in primary human chondrocytes. The Journal of biological chemistry PMID: 24469454