Epigenetic and microRNA regulation during osteoarthritis development

DNA methylation

DNA methylation is mediated by DNA methyltransferase (DNMT), which transfers the methyl group from the donor, methylated S-adenosyl-methionine (methyl-SAM), to DNA bases, particularly cytosine (CpG island). DNA methylation occurs in both the gene promoter region and gene bodies and regulates gene transcription^46–48. Recent studies found that DNA methylation is dynamically regulated through a cyclic enzymatic cascade composed of cytosine methylation by DNMTs and demethylation by ten-eleven translocation methylcytosine-(TET) dioxygenases (TET1, 2, and 3)⁴⁹. In mammals, there are three enzymatically active DNMTs, DNMT1, DNMT3a, and DNMT3b, and one related regulatory protein, DNMT3L⁴⁸. DNMT1 is primarily a “maintenance” methyltransferase that recognizes the hemi-methylated DNA strand and preserves the methylation pattern throughout cell replication and division. The global knockout of the Dnmt1 gene is embryonically lethal at E10.5 because of a significant loss of global DNA methylation, suggesting that DNA methylation is essential for normal mammalian development⁵⁰. In contrast, two de novo DNMTs, 3a and 3b, have tissue-specific expression patterns and create unique methylation signatures. Knockout mice with Dnmt3b deletion showed embryonic lethality between E11.5 and E15.5 as well as several skeletal defects, including growth impairment. However, loss of the Dnmt3b gene does not affect the entire genome methylation pattern⁵¹.

In recent decades, researchers have studied changes in the DNA methylation status of individual genes during OA development and progression and found that the promoter of Col10a1 appeared to be hypomethylated during chondrocyte hypertrophy and maturation followed by its upregulation⁵². Similarly, the CpG sites within the promoter area of a number of metalloproteinases, including MMP2, MMP9, MMP13, and ADAMTS4, showed decreased methylation profiles in OA compared to normal cartilage, correlating with elevated gene expression and resulting in ECM degradation^53,54. Reduced CpG methylation was reported in the MMP13, IL-1β, and inducible nitric oxide synthase (iNOS) promoter in OA tissue which correlates with the increased MMP13, IL-1β, and iNOS expression in OA chondrocytes^55,56. During the chondrocyte maturation process, changes in DNA methylation patterns were observed in several transcription factors, such as Sox9 and Runx2⁵⁷. Hypomethylation in promoter regions of those genes promoted gene transcription, which further activated downstream signaling molecules, including MMPs, and eventually stimulated chondrocytes toward hypertrophy and terminal maturation. Either hypomethylation or hypermethylation occurred in promoter regions within a subset of OA-specific genes, including ligands (e.g., BMP7 and IL-1β)^58,59, receptors, transcription factors (e.g., Sox9 and Runx2)⁵⁷, enzymes (e.g., MMPs and ADAMTS4/5)^53,54, and ECM proteins (e.g., aggrecan, Col2a1, and Col10a1)^52,60.

Recent methylome screening data further confirmed that alterations in DNA methylation occurred in OA chondrocytes and that chondrocyte transcriptomes may be changed in OA patients, indicating that DNMTs influence OA susceptibility and severity by modulating pathways or signals leading to OA^{16–18,61,62}. However, which DNMT factor or factors mediate these changes genome-wide remains largely unknown. In one of our ongoing experiments, we have found that DNMT3b, but not DNMT 1 or 3a, was highly expressed in articular chondrocytes, but its expression was significantly decreased in chondrocytes derived from patients with OA or from several OA mouse models, including the aging animal model, meniscal ligamentous injury (MLI) model, and obesity model (Shen et al., unpublished data). Recent reports demonstrated that TET1, 2, and 3 are present in human chondrocytes and that TET1 expression was significantly reduced by inflammatory factors, such as IL-1β or TNFα⁶³. Recent studies have also revealed a significant increase in 5-hydroxymethylcytosine levels in OA chondrocytes because of TET1 downregulation^64,65. Because DNA methylation is a reversible process, the role of the TET family members in OA development needs further investigation to better understand the regulation of DNA demethylation during OA development and progression.

The regulation of transcription factors on chondrocyte-specific genes through alterations of DNA methylation and histone modification has been reported in recent years. For example, it has been reported that methylation of the -110 bp CpG site in the Mmp13 promoter strongly correlates with the high Mmp13 expression in chondrocytes. This CpG site resides within a HIF consensus motif. The methylation of this site will decrease HIF-2α binding to the Mmp13 promoter⁵⁵. AT-rich interactive domain 5b (Arid5b) is a newly identified transcriptional co-regulator of Sox9. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrocyte marker genes, H3K9me2 levels are increased in Arid5b knockout chondrocytes⁶⁶.

Histone modification

Working closely with DNA methylation, histone modification—including acetylation, phosphorylation, methylation, and ubiquitination—regulates gene expression by controlling the accessibility of the transcriptional machinery^67,68. Recent studies demonstrated that histone acetylation and deacetylation are involved in OA pathogenesis by affecting chondrocyte anabolic and catabolic processes. Histone acetylation is mediated by histone acetyltransferases (HATs) and is a critical step in loosening the DNA structure, which allows regulatory factors to access the transcriptional machinery and the subsequent initiation of gene expression, whereas deacetylation is considered the termination or repression of gene expression⁶⁹. Histone deacetylation is mediated by histone deacetylases (HDACs), including the classic HDAC and NAD⁺-dependent silent information regulator 2 (SIR2) families^70,71. The use of large-scale analysis (ChIP-seq) of chondrocyte histone acetylation did not find global alterations in OA chondrocytes but did find changes in specific gene loci, encoding MMPs, ECM molecules, and inflammatory factors.

In patients with OA, elevated HDAC7 expression has been reported to contribute to cartilage degradation by inducing Mmp13 expression in OA cartilage. The inhibition of HDAC7 in vitro leads to suppression of inflammatory factor-induced Mmp13 expression⁷². The expressions of HDAC1 and HDAC2 are upregulated in OA synovial tissue as well, and this may lead to repression of Col2a1 expression in chondrocytes by interfering with the recruitment of Snail^73,74. Therefore, HDAC inhibitors have been extensively studied in various OA models. Specific HDAC inhibitors can inhibit cytokine-induced MMP expression in chondrocytes to protect against proteoglycan loss and cartilage degradation^75–77. HDAC inhibitors can also stimulate the expression of ECM components—such as Col2a1, cartilage oligomeric matrix protein (COMP), and aggrecan—in chondrocytes^74,78. In the rabbit anterior cruciate ligament transection (ACLT) model, an HDAC inhibitor significantly decelerated injury-induced cartilage erosion, mainly due to reduced expression of MMPs and inflammatory cytokines, indicating that HDAC inhibitors may provide a potential treatment for OA⁷⁹.

In the SIR2 family, SIRT1 has been extensively studied. SIRT1 is highly expressed in chondrocytes and its expression was found to be decreased in OA cartilage^80,81. SIRT1 can promote expression of ECM genes, such as Col2a1, Col9a1, and COMP, possibly through deacetylation of Sox9, while inhibiting Col10a1 and Adamts5⁸². SIRT1 also prevents apoptosis in chondrocytes by enhancing insulin-like growth factor (IGF) signaling to inactivate p53. The reduction of SIRT1 expression leads to an increase in chondrocyte apoptosis in OA cartilage⁸³. Interestingly, the function of SIRT1 is closely linked to the inflammatory response and the hypoxic response as well, although SIRT1 has not been approved for use to treat OA. In a variety of tissues, SIRT1 initiates a gene-specific transcriptional repression program to terminate inflammatory response by deacetylating the p65 subunit of nuclear factor-kappa-B (NF-κB) and blocking NF-κB binding to the DNA elements^84,85. SIRT1 can also directly deacetylate and activate HIF-2α, which is upregulated in OA cartilage, to promote MMP expression and eventually degrade the articular cartilage^86,87.

In addition to histone acetylation, histone H3K4 methylation mediated by histone-lysine N-methyltransferase (HMT) was recently investigated. HMT expression level was found elevated in OA cartilage, which resulted in H3K4 methylation at the iNOS and COX-2 promoter areas and induction of gene expression⁸⁸. Similarly, an age-dependent increase in H3K4me2 occurs in the nuclear factor of activated T cells 1 (Nfat1) promoter, which led to suppression of Nfat1 expression in adult articular chondrocytes and eventually developed OA-like phenotype in mice^89,90. Increased demethylation mediated by histone demethylase LSD1 was also found in OA chondrocytes. Elevated LSD1 contributed to H3K9 demethylation in the microsomal prostaglandin E synthase 1 (mPGES-1) promoter and induction of gene expression in human OA chondrocyte⁹¹. Moreover, the architecture of histone acetylation and methylation in local genome can further guide the long-range chromatin interaction to regulate specific gene regulatory DNA elements⁹².

The role of miR-140 in osteoarthritis pathogenesis

miRNAs are endogenous non-coding RNAs and play important roles in negative regulation of RNA stability and protein expression^93,94. Several miRNAs have been found to be more abundant in articular chondrocytes than in undifferentiated mesenchymal stem cells. The best example of this is miR-140⁹⁵. miR-140 is found in an intron of the Wwp2 gene coding for WWP2 E3 ubiquitin ligase⁹⁶. Deletion of miR-140 did not alter the expression level of Wwp2 in chondrocytes⁹⁷. Analysis of the intronic sequence found two miR-140s: miR-140-5p and miR-140-3p⁹⁸. The expression levels of miR-140-5p and -3p were both significantly reduced in OA chondrocytes⁹⁸. During chondrocyte differentiation, miR-140 expression increased in parallel with Sox9 and Col2a1. However, in OA tissues, miR-140 expression is reduced and Adamts5 expression was upregulated⁹⁵. In vitro treatment of chondrocytes with IL-1β suppresses miR-140 expression⁹⁵. miR-140 is the only miRNA with a cartilage-specific expression pattern^95,99. miR-140 deficiency accelerates chondrocyte differentiation into hypertrophic chondrocytes and inhibits differentiation of resting chondrocytes into columnar proliferating chondrocytes¹⁰⁰. The reduction in miR-140 expression in OA cartilage may contribute to abnormal gene expression during OA development⁹⁵. For example, miR-140 regulates the expression of histone deacetylase 4 (HDAC4), a co-repressor of Runx2 and myocyte-specific enhancer factor 2 (Mef2)¹⁰¹. miR-140 also targets Cxcl12⁹⁹ and Smad3¹⁰², both of which are implicated in chondrocyte differentiation. In miR-140 null mice, OA-like changes were observed and characterized by proteoglycan loss and fibrillation of articular cartilage, probably due to increased Adamts5 expression¹⁰³. This increased Adamts5 expression was reversed by transfection of ds-miR-140 into miR-140-deficient chondrocytes¹⁰³. In addition, cartilage-specific miR-140-overexpressing transgenic mice had no abnormal skeletal phenotype during embryonic development but did show a protective effect in an antigen-induced arthritis model¹⁰³. However, the upregulation of Adamts5 and Hdac4 expression in chondrocytes was not found in the other miR-140 knockout mouse model generated by Nakamura et al.⁹⁷. Instead of upregulation of Hdac4 expression, miR-140 enhances HDAC4 function in chondrocytes¹⁰⁰. miR-140 could interact with PTHrP-HDAC4 pathway to control chondrocyte differentiation. miR-140 deficiency and PTHrP or Hdac4 heterozygosity synergistically impair skeletal growth. Loss of miR-140 upregulates MEF2C expression. miR-140 negatively regulates p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduces MEF2C expression¹⁰⁴. The functional role of miR-140 in cartilage homeostasis is also involved in the regulation of MMP13¹⁰⁵. MMP13 is a well-known key player in cartilage biology and OA pathology. It has been reported that miR-140 is a negative feedback regulator of MMP13¹⁰⁶. In addition, transfection with pre-miR-140 significantly decreased IGFBP-5 expression. In contrast, transfection with anti-miR-140 significantly increased IGFBP-5 expression¹⁰⁷.

The role of Runx2 in osteoarthritis development

Significant progress has been made in recent years in OA research, and several OA mouse models, including genetic models and surgically induced OA models, have been developed and reported. One common feature of these animal models is upregulation of Runx2^5,9,36, leading to further increases in genes coding for matrix degradation enzymes, such as Mmp9, Mmp13, and Adamts5, because Runx2 is a key transcription factor regulating the transcription of these genes^108–110. Key questions are how Runx2 is regulated and whether a therapeutic strategy can be developed by downregulation of Runx2 in OA cartilage.

During skeletal development, Runx2 mRNA expression was detected in skeletal elements as early as E10.5 and E11.5; however, hypertrophic chondrocytes and primary ossification centers do not form until E14.5, although Runx2 is a key transcription factor driving chondrocyte hypertrophy¹¹¹. These findings suggest that Runx2 protein expression is suppressed because of post-transcriptional regulation during early skeletal development since chondrocyte proliferation and expansion are needed at this stage. These findings also suggest that there is an endogenous negative regulatory mechanism for Runx2 protein expression.

MicroRNA regulation of Runx2 expression

In recent studies, we have examined potential miRNAs that may bind the 3′-non-coding region of the Runx2 gene and found that miR-204 and miR-211, two homologous miRNAs, bind Runx2 and regulate Runx2 expression in mesenchymal progenitor cells¹¹². To further investigate the functions of these miRNAs in the regulation of Runx2 protein expression in articular chondrocytes and in cartilage homeostasis, chondrocyte-specific miR-204 and miR-211 transgenic mice and conditional knockout mice need to be generated and tested. In addition to miR-204 and miR-211, several other miRNAs have been reported to regulate Runx2 expression¹¹³. Their functions in OA development also need further investigation.

The role of miRNA regulation in OA development involves upstream regulation and downstream gene targeting. For example, it has been reported that IL-1β, an inflammatory cytokine, suppresses the expression of miR-140, which in turn causes upregulation of Adamts5, a target gene of miR-140, in chondrocytes^95,103, so miR-140 could serve as a mediator during OA development. In addition, it has been reported that TGF-β/Smad3 regulates miR-140 expression in OA chondrocytes¹¹⁴. TGF-β signaling is one of the key signaling pathways in OA development and responds to mechanical loading. Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel post-transcriptional regulator of IL-6 expression and is targeted by miR-9. MCPIP-1 mRNA expression was low, but expression of miR-9 and IL-6 was high, in damaged OA cartilage. MCPIP-1 protein directly binds with IL-6 mRNA, and overexpression of wild-type MCPIP-1 destabilized the IL-6 mRNA. MCPIP-1 expression was altered by overexpression or inhibition of miR-9. These findings implicate miR-9-mediated suppression of MCPIP-1 in the pathogenesis of OA via upregulation of IL-6 expression in IL-1β-stimulated human OA chondrocytes¹¹⁵. These studies also suggest that miRNAs may serve as important mediators in OA, although they may not be able to trigger the OA occurrence.