Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis

Histone modification

The post-translational modification of histones includes acetylation, methylation, ubiquitination, sumoylation, and phosphorylation. The overall pattern of histone modification regulates interaction between DNA, regulatory proteins, and other histone molecules, thereby affecting gene expression (Kouzarides, 2007).

Histone N-terminal tails protruding from nucleosomes are subjected to acetylation modifications by histone acetyltransferases (HATs), which include three major families: Gcn5 N-acetyltransferases (GNATs), p300/CBP, and MYST family (named for its founder members Morf, Ybf2, Sas2, and Tip60) (Sterner and Berger, 2000). Histone acetylation mediated by p300/CBP leads to transcriptional activation of SOX9, illustrating the role of the HAT family in sustaining chondrocyte homeostasis (Furumatsu and Asahara, 2010; Furumatsu et al., 2005).

Histone deacetylases (HDACs), in contrast, oppose the action of HAT enzymes by removing the acetyl group from lysine residues. HDACs are subdivided into two groups: The SIR2 family of NAD⁺-dependent protein deacetylases and the classical HDAC family. The SIR2 family, consisting of seven family members in mammals (SIRT1-7), removes acetyl groups from lysine residues of histones H3 and H4, and non-histone proteins (Blander and Guarente, 2004; de Ruijter et al., 2003). In chondrocytes, SIRT1, one of the most extensively studied SIRT family members, was shown to promote the expression of cartilage-specific genes in a SOX9-dependent manner (Dvir-Ginzberg et al., 2008). SIRT1 forms a complex with SOX9 and targets it for deacetylation. The complex is subsequently brought to the enhancer and promoter regions of COL2A1, where it further recruits coactivators such as GCN5, PGC1α, and p300, modulating the acetylation state of H3K9/K14 and H4K5, and mediating tri-methylation of H3K4 (Dvir-Ginzberg et al., 2008). In line with reports revealing the anabolic roles of SIRT1 in chondrocytes, other chondroprotective roles of SIRT1 have been noted in the context of OA pathogenesis. SIRT1 suppresses protein tyrosine phosphatase 1B (PTP1B) and activates insulin-like growth factor (IGF) receptor pathway, enhancing survival of chondrocytes (Gagarina et al., 2010). Moreover, TNFα signaling triggers cathepsin B-mediated cleavage of SIRT1, producing 75-kDa SIRT1 (Dvir-Ginzberg et al., 2011). This 75-kDa SIRT1 has been shown to be implicated in protecting chondrocytes from apoptosis following exposure to proinflammatory cytokines (Oppenheimer et al., 2012). Consistently, heterozygous haploinsufficient Sirt1^+/− or Sirt1 mutant mice lacking SIRT1 enzymatic activity show increased chondrocyte apoptosis and enhanced OA severity with age (Gabay et al., 2012; 2013; Matsuzaki et al., 2014). Furthermore, the expression level of SIRT1 is reduced over the course of OA, giving rise to hypertrophic phenotypes and OA-like gene expression patterns in chondrocytes (Fujita et al., 2011). Similarly, surgically-induced OA pathogenesis in mice was significantly accelerated by chondrocyte-specific deletion of the Sirt1 gene, with concomitant downregulation of anabolic proteins and upregulation of cartilage degrading enzymes (Gabay et al., 2013; Matsuzaki et al., 2014). Moreover, in Sirt1 CKO mice, p65 was found to be acetylated, which in turn causes activation of NF-κB pathway and consequently catabolic pathways, augmenting OA pathogenesis (Fig. 1) (Dvir-Ginzberg et al., 2011). Additionally, SIRT6 has been shown to be responsible for controlling senescence in cartilage by protecting chondrocytes from DNA damage, telomere dysfunction, and premature senescence (Nagai et al., 2015).

Classical HDACs remove acetyl groups from N-acetyl lysine residues on histones, enabling the histones to wrap DNA more tightly to repress transcription. The broad inhibition of HDACs by trichostatin A (TSA) or PXD101 was shown to suppress COL2A1 expression in chondrocytes, suggesting that the HDAC family plays a role in regulating chondrocyte anabolism. Moreover, HDAC inhibition promotes acetylation of the WNT-5A promoter, which in turn leads to transcriptional suppression of COL2A1 (Huh et al., 2007). HDAC1 and HDAC2 activity were found to be responsible for the transcriptional repression of cartilage-specific genes through the activation of NF-κB signaling (Hong et al., 2009; Zhong et al., 2002). HDAC4 was identified as a core element regulating hypertrophic maturation of chondrocytes and endochondral bone formation. HDAC4 not only suppresses the expression of RUNX2 at the transcriptional level via interaction with histones, but also interacts with RUNX2 directly, reducing its transcriptional activity (Vega et al., 2004). HDAC7, on the other hand, plays a role in the regulation of chondrocyte catabolism. HDAC7 activity is markedly upregulated in OA cartilage, and essentially required for IL-1β-mediated expression of MMP13 (Higashiyama et al., 2010).

HDACs generally augment imbalance between catabolism and anabolism in chondrocytes, and the potential of HDAC inhibitors as therapeutic agents for OA treatment have been extensively investigated. TSA, butyric acid (BA), and vorinostat treatment have been shown to effectively diminish IL-1-mediated expression of inducible nitric oxide (NO) synthase (iNOS), COX2, and MMPs (Chabane et al., 2008; Zhong et al., 2013). These anti-inflammatory effects of HDAC inhibitors were demonstrated in vivo, with concomitant suppression of osteoarthritic cartilage destruction and synovitis (Nasu et al., 2008).

Meanwhile, histone methylation is mediated by histone methyl transferases (HMTs). Methylation can occur on both lysine and arginine residues in histones without altering the net charge of the affected residues. SOX9, a transcriptional regulator of COL2A1 and ACAN, is itself subjected to progressive epigenetic regulation over the course of OA progression. Under OA-related pathogenic conditions, the promoter of SOX9 acquires a epigenetic profile characterized by elevated levels of trimethylation of H3K9 and H3K27, and decreased acetylation of H3K9, 15, 18, 23, and 27, which collectively leads to transcriptional repression of SOX9 (Kim et al., 2013). Histone-lysine N-methyltransferase SETD7 (SET7/9) forms a complex with SIRT1 in the COL2A1 promoter region and elevates trimethylation of H3K4, thereby enhancing COL2A1 expression (Oppenheimer et al., 2014). Nuclear factor of activated T-cells 1 (NFAT1) is a transcription factor whose deficiency is correlated with the onset of OA-related phenotypes (Wang et al., 2009). Age-dependent expression of NFAT1 is primarily regulated by histone demethylation. Demethylation at H3K4 by lysine-specific demethylase-1A (LSD1) appears to be associated with transcriptional activation of NFAT1 during cartilage development, whereas an aging-associated increase in H3K9 methylation due to reduced lysine-specific demethylase 3A (KDM3A) level is responsible for transcriptional repression of NFAT1 (Rodova et al., 2011).

Metabolites and obesity

Epigenetic modifications such as acetylation, methylation, and glycosylation are influenced by the intracellular metabolic state. By altering epigenetic profiles, cells respond to metabolic challenges and adapt to it by subsequently changing intracellular signal transduction and gene expression (Metallo and Vander Heiden, 2010).

Oxygen levels in the joint environment substantially affect energy and metabolic state of chondrocytes, influencing catabolic and anabolic factor expression (Schipani et al., 2001). In particular, in articular cartilage which is under hypoxic conditions due to its avascular nature, HIF-1α may serve as an epigenetic mediator by directly inducing SIRT1 expression and activating SIRT1-driven epigenetic programs (Chen et al., 2011).

Metabolites from cellular metabolic pathways may act as sensors of energy state, and mediate corresponding epigenetic changes. Acetyl-CoA, a component of the tricarboxylic acid (TCA) cycle and an essential building block for key metabolic biomolecules, can also serve as a substrate for HATs (Kaelin and McKnight, 2013). Consequently, the intracellular acetyl-CoA level determines the acetylation patterns of lysine residues as a measure of metabolic state, regulating cell growth and proliferation (Cai and Tu, 2011). Moreover, the activity of ATP-citrate lyase (ACL), which is an enzyme synthesizing acetyl-CoA from glucose-derived citrate in mammals, depends on glucose abundance. Therefore, ACL is capable of serving as a nutrient sensor, linking intracellular energy state into histone acetylation and gene expression profiles (Hatzivassiliou et al., 2005; Wellen et al., 2009).

Methyltransferases use S-adenosylmethionine (SAM), derived from the methionine cycle, as a methyl group donor. The production rate of SAM is regulated by the activity of ATP-dependent methionine adenosyltransferase (MAT). Thus, cellular ATP controls the intracellular level of SAM and the activity of various methyltransferases, in turn influencing transcription profiles (Nishikawa et al., 2015). Many other enzymes regulating epigenetic changes use biological metabolites such as NAD⁺ (protein deacetylase/mono-ADP ribosyltransferase, and sirtuin), FAD (FAD-dependent histone demethylase), and 2-oxoglutarate (2-oxoglutarate-dependent histone demethylase) as cofactors, or UDP-N-acetylglucosamine (O-linked N-acetylglucosamine transferase) as a substrate.

The effect of metabolites on epigenetic regulation supports the link between metabolism and the molecular pathogenesis of various diseases (Kaelin and McKnight, 2013). SIRT family members have been demonstrated to be key metabolic regulators in controlling glucose and lipid metabolism (Picard et al., 2004; Schwer and Verdin, 2008), and gluconeogenesis in muscle and liver (Liu et al., 2008). SIRT1 promotes insulin secretion and ATP production upon glucose stimulation of pancreatic β cells (Bordone et al., 2006). In metabolic syndrome patients with insulin resistance or atherosclerosis, downregulation of SIRT1 was observed in monocytes in response to glucose or fatty acid treatment (de Kreutzenberg et al., 2010). Isoforms of the HDAC family are implicated in regulating lipid metabolism during adipogenesis (Iyer et al., 2012). Genetic deletion of Hdac1 and Hdac2 in mesenchymal precursor cells results in reduced adipogenesis and adipocyte differentiation, (Haberland et al., 2010), while Hdac9 knockout mice exhibited accelerated adipogenic differentiation (Chatterjee et al., 2011). Protein demethylase KDM3A is coupled to the regulation of genes involved in beta-adrenergic stimulated glycerol release and fatty acid oxidation such as uncoupling protein 1 (UCP1) (Tateishi et al., 2009). Extensive correlation between epigenetics and abnormal metabolic state have been noted in metabolic diseases such as obesity (Campion et al., 2009) and type 2 diabetes (Campion et al., 2009; Ling and Groop, 2009).

There has been substantial evidence for the idea that the prevalence of OA is positively correlated with that of metabolic syndrome (Table 1) (Kornaat et al., 2009; Puenpatom and Victor, 2009; Zhuo et al., 2012). Among the main features of metabolic syndrome, obesity has been considered to be a major risk factor for OA occurrence in the joints of knees, hip, and hands (Griffin and Guilak, 2005; Oliveria et al., 1999). However, a mechanistic understanding of the association between obesity and OA remains elusive. The simplest view indicates that obesity exacerbates joint strength by imposing a higher degree of mechanical loading on the joint (Guilak, 2011). Clinical studies have supported the idea that abnormal loading jeopardizes the structure and integrity of the knee joint, facilitating OA development (Maly et al., 2005; Mundermann et al., 2005). However, the notion that mechanical loading is the sole factor for obesity-induced OA is controversial. A series of studies demonstrate that the incidence and severity of hand OA have a positive correlation with body mass index (BMI), although mechanical loading from body weight cannot participate in the pathogenesis of this type of OA (Carman et al., 1994).

Adipokines released from fat tissues may represent a potential link between obesity and OA. Strikingly, obese mice with impairment in leptin signaling, due to deletion of leptin or leptin receptor, do not exhibit differences in terms of severity of OA in comparison to mice with normal body weight (Griffin et al., 2009). In fact, synovial fluid of OA patients was characterized to have elevated levels of leptin (Dumond et al., 2003; Ku et al., 2009). Increased levels of leptin in synovial fluid and cartilage promote cartilage catabolism by upregulating MMP13 and NOS2 expression (Iliopoulos et al., 2007; Otero et al., 2005).

Other adipokines, such as adiponectin (Chen et al., 2006; Lago et al., 2008) and visfatin, (Duan et al., 2012) were found to be responsible for the regulation of chondrocyte metabolism. Adiponectin is known to regulate homeostasis of whole body energy by controlling glucose and lipid metabolism, and abnormalities in its regulation have been implicated in several metabolic disorders including obesity (Kim et al., 2015). While alterations of adiponectin level were noted in OA cartilage and synovial fluid (Honsawek and Chayanupatkul, 2010; Koskinen et al., 2011; Yusuf et al., 2011), the role of this adipokine in OA development remains controversial. A study by Kang and colleagues suggests that that adiponectin promotes chondrocyte catabolism by upregulating MMPs and NOS2 levels via AMPK and JNK pathways (Kang et al., 2010). Others have suggested a chondroprotective function for adiponectin, demonstrating its role in upregulating inhibition of metalloproteinase 2 (TIMP2) and downregulating MMP13 (Chen et al., 2006; Yusuf et al., 2011). Visfatin (encoded by NAMPT) is an adipokine that has been found to be upregulated in chondrocytes, synovial fluid, and synoviocytes on OA. Visfatin inhibits matrix proteoglycan synthesis and increases the expression of matrix-degradative enzymes (Duan et al., 2012; Gosset et al., 2008; Yammani and Loeser, 2012; Yang et al., 2015). Notably, visfatin can be localized intracellularly, serving as a regulator of salvage pathways of NAD⁺ synthesis. Increased level of NAD⁺ by intracellular visfatin enhances enzymatic activity of SIRT1, affecting the expression of SOX9 and type II collagen through epigenetic regulation (Hong et al., 2011).

In addition to obese individuals, patients with hypertension are highly likely to suffer from OA (Conaghan et al., 2005; Hart et al., 1995). Hypertension triggers the onset of subchondral ischemia, which is considered to promote the pathogenesis of OA, by restricting the formation of subchondral vessels and limiting nutrient and gas support to cartilage (Findlay, 2007). Hypertension is presumed to affect a subset of epigenetic pathways that are regulated by glucose or oxygen in cartilage.