microRNAs implicated in chondrogenesis
The hallmark of osteoarthritis (OA) is the progressive degeneration of articular cartilage, although bone and synovia are also involved in the development of the disease . Chondrocytes are the unique cellular component of articular cartilage and, under physiological conditions, are responsible for a subtle balance between the synthesis of extracellular matrix (ECM) components, mainly type-II collagen and aggrecan , and its degradation by proteolytic enzymes such as the matrix metalloproteinases (MMP)  and A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) . In OA, there is an imbalance of this process driven by cytokines and the production of inflammatory mediators, resulting in an increase of the degradation process with respect to synthesis, and leading to articular cartilage loss [1, 5].
OA is considered a multifactorial disease in which genetics and environmental factors, such as aging, gender and obesity, among others, are strongly related with its development . Primary OA possesses an important genetic component, and several genetic association studies have demonstrated that it is associated with different genes that encode molecules involved in a number of pathways, such as inflammation, Wnt signalling, bone morphogenetic proteins (BMPs), proteases and their inhibitors, and extracellular matrix proteins, among others [7, 8]. However, there has not always been consistency in the results, probably due to the low penetrance of the gene polymorphisms studied, or to different gene-gene interactions and gene-environment interactions. In this regard, epigenetics is one mechanism through which gene-environment interactions occur. Epigenetics refers to heritable changes in gene expression that occur without changes in DNA, and includes DNA methylation, histone modifications, chromatin remodelling and microRNAs (miRNAs), although debate continues concerning whether miRNA can be categorized as an epigenetic phenomenon . Recent evidence has made it apparent that epigenetic changes alter the expression of genes that could participate in the pathogenesis of OA. This chapter does not intend to conduct a deep review of epigenetic modifications, but rather to review the main findings directly related with OA.
2. DNA methylation in osteoarthritis
DNA methylation is the most studied epigenetic mark in humans. DNA methylation involves the addition of a methyl group at CpG dinucleotides to convert cytosine into 5-methylcytosine. These CpG dinucleotides tend to cluster in regions termed ‘islands’, and approximately 70% of human gene promoters are associated with CpG, which are usually unmethylated. CpG-island methylation is associated with gene silencing due to the binding of methyl-CpG-binding proteins, which recruit proteins to the gene promoter, blocking its transcription. DNA methylation can also occur in CpG island shores, regions of lower CpG density that lie in close proximity to CpG islands (~2 kb); their methylation is associated with transcriptional inactivation. DNA methylation is mediated by the DNA methyltransferases (DNMTs) family of enzymes that catalyse the transfer of a methyl group to DNA. In mammals, five members of the DNMT family have been reported: DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L, but only DNMT1, DNMT3a and DNMT3b possess methyltransferase activity. DNMTs are classified as
Analysis of the overall methylation level of genomic DNA in the chondrocytes of degenerated OA cartilage shows no difference in comparison with normal chondrocytes. However, an inhibitor of cell proliferation, the cyclin-dependent kinase inhibitor 1 (
In chick embryos’ chondrocytes, the
The nuclear transcription factor SOX9, along with other SOX family members, is required for the control of the expression of ECM components. SOX9 is an important regulator of the chondrocyte phenotype and controls the expression of the
Metalloproteinase expression in normal cartilage is relatively low but is elevated in OA, resulting in ECM degradation [3, 23]. The altered synthesis of the cartilage-degrading enzymes in OA is the result of changes in the methylation status, as demonstrated by the analysis of the methylation of the promoter region of
Nitric oxide (NO) is a multifunctional molecule that suppresses energy production by mitochondrial respiration. In OA, high amounts of NO are produced, a consequence of upregulation in the chondrocyte of the inducible NO synthase (iNOS) induced by inflammatory cytokines, such as interleukin 1-beta (IL-1β) and tumour necrosis factor alpha (TNFα), among others . NO suppresses the synthesis of the cartilaginous matrix . In culture, under-stimulated chondrocytes produce
To date, it is well recognized that OA has an important inflammatory component in its development mediated by proinflammatory cytokines, such as IL-1β and TNFα [2, 32]. Healthy chondrocytes do not express
Bone morphogenetic protein-7 (BMP-7) – or osteogenic protein-1(OP-1) – is one of the most potent growth factors for cartilage maintenance and repair. It possesses a critical role in human cartilage homeostasis, regulating numerous metabolic pathways that are not only limited to its well-documented anabolic function but also to its anti-catabolic activity . There is a positive correlation between age and the methylation of the
The growth differentiation factor 5 (
Methylation analysis of 23,367 sites (corresponding to 13,463 genes) through a genome-wide methylation profile of bone from patients with OA and osteoporosis (OP) revealed an inverse relationship between methylation and gene expression in both groups, with 271 CpG sites being less methylated in OP than in OA.
3. Histone modification, chromatin remodelling and osteoarthritis
DNA is compacted by the tight weaving of approximately 147 base pairs around the proteins’ denominated histones, forming a DNA-protein complex termed a ‘nucleosome’, the basic unit of chromatin. Each nucleosome consists of an octamer of two copies of the following four core histones: H2A, H2B, H3 and H4. The nucleosomes comprise a barrier to transcription that blocks the access of activators and transcription factors to their sites in DNA. The histones are subject to post-transcriptional modification; the most common are acetylation and methylation, although other modifications have been identified, including phosphorylation, ubiquitination, SUMOylation, citrullination, and adenosine diphosphate ribosylation. Histones acetylation occurs at lysine residues and is associated with DNA accessibility and transcriptional activity, whereas deacetylation is associated with transcriptional repression. Histone acetyltransferases (HATs) are enzymes that transfer the acetyl group onto the ε-amino group of the lysine residues within a histone tail; this is a reversible process, and the enzymes that remove the acetyl groups are known as ‘histone deacetylases’ (HDACs). Classical isoforms of HDACs comprise a total of 11, and are broadly divided into two classes: HDACs 1, -2, -3 and -8 are Class I HDACs, while Class II encompasses HDAC isoforms 4, -5, -6, -7, -9, -10 and -11. The newly characterized SIR2 family of HDACs (sirtuins), termed ‘Class III’, operate through a nicotinamide adenine dinucleotide (NAD+)-dependent mechanism. Histone methylation is another major modification that takes place in the ε-amino group of lysine residues; it is mediated by histone H3 N-lysine lysine methyltransferases (HKMTs). The effect of histone lysine methylation on gene regulation is highly complex, mediating either transcription repression or activation. Likewise, the methylation of arginine residues is catalysed by the protein arginine methyltransferase (PRMT) family. Regulation of gene transcription can also occur by chromatin remodelling. The SWItch/sucrose non-fermentable protein complex binds to the nucleosome and disconnects the DNA from the histones, creating a transient DNA loop and resulting in nucleosome repositioning, such that the transcription of targeted genes can be increased or decreased depending upon whether the gene is located in the open-chromatin or compacted chromatin region. Polycomb-group proteins are also involved in gene silencing through chromatin remodelling, repressing transcription by maintaining a heterochromatin state through particular histone modifications and DNA methylation [9-13].
Chondrocyte differentiation is controlled by transcription factors such as SOX9, among others. SOX9 requires other cofactors, such as the CREB binding protein, which activates SOX9-dependent transcription due to its intrinsic histone acetyl-transferase activity . In human chondrocytes inducted by IL-1β and the fibroblast growth factor 2 (FGF2), there is increased expression of MMPs and ADAMTS, responsible for collagen and aggrecan loss, respectively. However, HDAC inhibitors (HDACi) block the induction of these enzymes at the mRNA and protein levels [46, 47]. Thus, HDACi also suppress IL-1β-induced NO and prostaglandin E2 (PGE2) synthesis, which plays an important role in OA as well as in proteoglycan degradation .
Specific HDACs appear to be involved in different processes as well as targeting different chondrocyte-specific genes. In a murine model,
The role of HDACi has been explored in OA. HDAC activity decreases during chondrocyte dedifferentiation, and the inhibition of HDAC with HDACi trichostatin suppresses type II collagen expression. This is because HDACi promotes the acetylation of Wnt-5a, increasing its expression, which is known to inhibit type II collagen . Human chondrocytes under mechanical stress exhibit the downregulation of
Histone H3 lysine-4 (H3K4) methylation is associated with transcriptional activation, whereas H3K9 methylation correlates with transcriptional repression. In human osteoarthritic chondrocytes, the induction of iNOS and cyclooxygenase 2 (COX2) expressions by IL-1β are associated with H3K4 di and trimethylation at the
NAD-dependent class III HDACs consist of SIRT1-7. SIRT1 plays a key role in the regulation of metabolism, as well in regulating cell differentiation, proliferation, survival and longevity [60, 61]. SIRT1 increases cartilage-specific gene expression, such as
To date, OA is well-recognized as an inflammatory disease in which inflammatory cytokines play a central role. In human OA chondrocytes treated with TNFα there is an impaired SIRT1 activity due to cleavage mediated by cathepsin B, resulting in the upregulation of
4. microRNAs in chondrogenesis and osteoarthritis
MicroRNA (miRNA) are small noncoding RNAs of ~20–25 nucleotides (nt) in length that are transcribed in the nucleus by RNA polymerase II or III into a long precursor denominated primary-miRNA (pri-miRNA). This pri-miRNA is processed by the microprocessor Drosha-DGCR8 complex, an RNase III-type enzyme, to generate a precursor of ~70–100 nt, known as ‘pre-miRNA’, which is translocated into the cytoplasm via exportin 5. Pre-miRNA is processed by the ribonuclease dicer, generating a miRNA duplex of ~22 nt. Finally, one of the strands is incorporated into the RNA-induced silencing complex (RISC), where it is guided to its target mRNA. miRNA is involved in post-transcriptional gene-expression regulation, targeting 30% of the encoding genes through complementary base-pairing between the miRNA and the 3´-UTR of the messenger RNA (mRNA) target, resulting in the translational suppression or direct degradation of the mRNA [9-13].
4.1. Cartilage and miRNA
The influence of miRNAs in cartilage homeostasis and skeletal development has been demonstrated in recent years. Dicer is an essential enzyme for the generation of mature miRNA and for proper skeletal morphogenesis. In mouse models, the loss of
miR-140 is the most studied miRNA in both cartilage and OA. This miRNA was originally identified as a cartilage-restricted miRNA in developing zebra fish, with its expression in the jaw, head and fins during embryonic development . In mice, miR-140 is also expressed in cartilage during embryonic long- and flat-bone development . In a murine model with a targeted deletion of miR-140, mice are borne with grossly normal skeletal development; however, postnatally they manifest skeletal deformities with short stature and craniofacial deformities, probably as a result of abnormal chondrocyte proliferation [75, 76]. miR-140 is encoded in an intronic region of the ubiquitin E3 ligase gene,
In the gene expression pattern in human articular chondrocytes and human MSC, miR-140 saw the largest differences in expression. During chondrogenesis, miR-140 increases in parallel with the expression of
In addition to miR-140, miR-455-3p expression is also restricted to the cartilage and perichondrium of the developing long bones in chicks and to the long bones and joints in mouse embryos, and it contributes to chondrogenesis in humans. miR-455-3p resides in an intron of
To study the miRNA-mediated regulation of chondrogenesis, the expression of 35 miRNAs in chondroblasts derived from MSC was analysed and it was found that miR-199a and miR-124a were strongly upregulated, while miR-96 was substantially suppressed. The potential targets of the miRNAs are the following transcriptional factors: HIF-1α for miR-199a, regulatory factor X1 (RFX1) for miR-124a, and SOX5 for miR-96, which demonstrate that miRNAs and transcription factors could fine-tune cellular differentiation .
In another miRNA microarray in MSC at four different stages of TGF-β3-induced chondrogenesis differentiation, eight significantly upregulated and five downregulated miRNA were observed. Two miRNA clusters, miR-143/145 and miR-132/212, were maintained on downregulation, while miR-140-3p was the most upregulated. By means of bioinformatics approaches, the following target genes were predicted:
miR-675 could also regulate
A comparative miRNA array of approximately 380 miRNAs in C2C12 cells induced by BMP2 found that several miRNAs, including let-7e, miR-221, miR-199a-3p, miR-374 and miR-298 were positively regulated, while miR-125a, miR-210, miR-125b, miR-21, miR-145 and miR-143 were repressed. Among these, miR-199a-3p was the most significantly upregulated at 24 h following BMP2 induction in the C3H10T1/2 cells and in an
To investigate the miRNA expression pattern involved in the chondrocyte dedifferentiation process, a microarray analysis was performed. Several miRNA were deregulated, including 13 upregulated and 12 downregulated miRNA in differentiated as compared with dedifferentiated chondrocytes. The most notable changes were for miR-491-3p, miR-140-3p, miR-140-5p and let-7d, which were upregulated, and for miR-548e, miR-342-5p, miR-1248 and miR-146a, which were downregulated. Bioinformatics analysis revealed 21 microRNA–gene target pairs potentially involved in chondrocyte dedifferentiation. Among these, miR-548e-
miR-194 decreased during chondrogenic differentiation in adipose-derived stem cells (ASCs) (which are capable of differentiating into cartilage lineages
During the chondrogenesis differentiation of chick-limb mesenchymal cells, miR-221 may be involved in chondrocyte apoptosis; its inhibition reverses the chondro-inhibitory actions of a Jun N-terminal protein kinase (JNK) inhibitor in the proliferation and migration of chondrogenic progenitors. A target for miR-221 is Mmd2, an oncoprotein that has been shown to inhibit the activity of the p53 tumour suppressor protein with E3 ubiquitin ligase activity, and downregulation of Mmd2 prevents the degradation of the slug protein, which negatively regulates the proliferation of chondroprogenitors. The slug protein is a snail family member – it controls the developmental process by regulating the genes involved in cell adhesion and migration . In the same model, miR-34a is a negative modulator of chondrogenesis and affects MSC migration but not proliferation. EphA5, a receptor in Eph/ephrin signalling that mediates cell-to-cell interaction, is a miR-34a target . Moreover, miR-34a regulates RhoA/Rac1 cross-talk and negatively modulates the reorganization of the actin cytoskeleton, which is one of the essential processes in establishing chondrocyte morphology . Table 1 depicts the miRNAs implicated in chondrogenesis.
||Histone Deacetylase 4
Transcription Factor Sp1
A Disintegrin-Like And Metalloproteinase With Thrombospondin Type 1 Motif, 5
Chemokine, Cxc Motif, Ligand 12
Mothers against decapentaplegic homolog 3
||SRY-Related HMG-Box gene 5||83|
||Hypoxia-inducible factor 1-alpha||83|
||Regulatory factor x 1||83|
||Mothers against decapentaplegic homolog 1
Mothers against decapentaplegic homolog 5
||p53-binding protein Mdm||93|
||Type II collagen||87|
||Activin a receptor, Type IB||84|
||SRY-Related HMG-Box gene 6||84|
||Runt-related transcription factor 2||84|
||SRY-Related HMG-Box gene 9||85,86|
||Integrin, beta 3||89|
||SRY-Related HMG-Box gene 9||89|
||Insulin-Like Growth Factor-Binding Protein 5||89|
||Type II collagen||89|
||Integrin, beta 3||89|
||SRY-Related HMG-Box gene 5||89|
||Insulin-Like Growth Factor I||89|
||A Disintegrin And Metalloproteinase Domain 9||95|
||Activin a receptor, Type IIB
||Transforming Growth Factor-Beta Receptor, Type II||92|
||Matrix Metalloproteinase 13||96|
4.2. Osteoarthritis and miRNAs
The effects of miRNA deregulation on OA are evident in studies comparing the expression of miRNAs between OA tissue specimens and their normal articular counterparts. A study tested 365 miRNA expression in articular cartilage obtained from patients with OA undergoing total joint replacement surgery as well as from normal controls. The study identified 16 miRNA differentially expressed in OA cartilage, of which nine were upregulated and seven downregulated. Through an
Some miRNAs were examined as potential biomarkers of patients with OA of the knee, and miR-16, miR-132, miR-146a and miR-22 were significantly lower in the synovial fluid than in plasma. miR-132 in plasma exhibited a number of miRNAs in plasma, some of which were found at different levels between patients with rheumatoid arthritis (RA) and with OA. Concentrations of miR-16, miR-132, miR-146a and miR-223 are reduced in the synovial fluid of individuals suffering from OA compared with healthy controls . More recently a profiling of 384 miRNAs was developed in the plasma of patients with radiographic OA of the knee, and 12 miRNAs were found to be differentially expressed with a clear differentiation of OA samples from those of healthy controls.
miR-140 has also been implicated in OA pathogenesis. In mice, the disruption of miR-140
In human OA chondrocytes, miR-27a expression is decreased in OA and treatment with anti-miR-27a increases the expression of
Members of the miR-34 family are induced by p53, leading to apoptosis, cell cycle arrest and senescence through targeting E2F3, cyclin E2 and CDK6, etc. . miR-34a is the most significantly induced miRNA after the activation of p53. In chondrocytes of a rat model of OA, miR-34a is upregulated after IL-1β stimulation and its silencing prevents the IL-1β-induced upregulation of iNOS and the downregulation of Col2a1 .
miR-146a/b has been described as a key molecule in the inflammatory response  and is expressed in all layers of human articular cartilage, especially in the superficial zone. In grade I OA, the expression of miR-146a and COL2A1 is significantly increased, and is decreased in grades II and III, along with MMP13. Thus, their expression gradually decreased with progressive tissue degeneration . In a rat model with surgically induced OA, miR-146a is upregulated in articular chondrocytes in response to treatment with IL-1β.
||A Disintegrin-Like And Metalloproteinase With Thrombospondin Type 1 Motif, 5
Matrix Metalloproteinase 13
Insulin-Like Growth Factor-Binding Protein 5
||Interleukin 1 Receptor-Associated Kinase 1
TNF Receptor-Associated Factor 6
||Matrix Metalloproteinase 13
Mothers against decapentaplegic homolog 4
||AcyL-CoA Oxidase 1||97|
||Tissue Inhibitor Of Metalloproteinase 2||97|
||Peroxisome Proliferator-Activated Receptor
Bone Morphogenetic Protein 7
||Growth/Differentiation Factor 5||97|
||Integrin, Alpha 5||97|
||TNF Receptor-Associated Factor 4||97|
||SRY-Related HMG-Box gene 9||97|
||Matrix Metalloproteinase 13||98|
||Matrix Metalloproteinase 13
Insulin-Like Growth Factor-Binding Protein 5
||Matrix Metalloproteinase 13||104|
||Histone Deacetylase 4||111|
||Solute carrier family 39 (zinc transporter), member 8||112|
At present, there is increasing progress in the description of epigenetic mechanisms under normal conditions, as well in disease, and much of the current knowledge has focused on cancer. Yet epigenetics research has provided new insights in relation to other entities, such as neurological and autoimmune diseases. Epigenetics research into OA continues to be developed, but could shed light on its pathological mechanisms. One promising field is related with OA treatment, such as HDACi or miRNAs. However, although HDACi and miRNAs could inhibit several genes related with its development, they also inhibit ECM genes. To date, there is no appropriate biomarker for OA. Epigenetics marks in OA have been associated with the condition, and even with its progression, and could be biomarkers of disease and progression, as miRNAs determined in plasma. However, research into epigenetics continues to be required.
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), proyecto SALUD-2012-C01-180720.
Goldring MB, Goldring SR. Articular cartilage and subchondral bone in the pathogenesis of osteoarthritis. Ann N Y Acad Sci 2010; 1192: 230-7.
Maldonado M, Nam J. The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int 2013; 2013: 284-873. doi: 10.1155/2013/284873.
Knapinska A, Fields GB. Chemical biology for understanding matrix metalloproteinase function. Chembiochem 2012; 13(14): 2002-20.
Verma P, Dalal K. ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis. J Cell Biochem. 2011; 112(12): 3507-14.
Houard X, Goldring MB, Berenbaum F. Homeostatic mechanisms in articular cartilage and role of inflammation in osteoarthritis. Curr Rheumatol Rep 2013; 15(11): 375.
Johnson VL, Hunter DJ. The epidemiology of osteoarthritis. Best Pract Res Clin Rheumatol. 2014; 28(1): 5-15.
Valdes AM, Spector TD. The contribution of genes to osteoarthritis. Rheum Dis Clin North Am 2008; 34(3): 581-603.
Reynard LN, Loughlin J. The genetics and functional analysis of primary osteoarthritis susceptibility. Expert Rev Mol Med. 2013; 15: e2.
Tammen SA, Friso S, Choi SW. Epigenetics: the link between nature and nurture. Mol Aspects Med. 2013; 34(4): 753-64.
Kim JK, Samaranayake M, Pradhan S. Epigenetic mechanisms in mammals. Cell Mol Life Sci 2009; 66(4): 596-612.
Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010; 28(10): 1057-68.
Golbabapour S, Abdulla MA, Hajrezaei M. A concise review on epigenetic regulation: insight into molecular mechanisms Int J Mol Sci. 2011; 12(12): 8661-94.
Walters DM. Epigenetic considerations in medicine. N C Med J 2013; 74(6): 534-6.
Fang JY, Lu YY. Effects of histone acetylation and DNA methylation on p21(WAF1) regulation. World J Gastroenterol 2002; 8(3): 400-5.
Sesselmann S, Söder S, Voigt R, Haag J, Grogan SP, Aigner. DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes. Osteoarthritis Cartilage 2009; 17(4): 507–512.
Fernandez MP, Young MF, Sobel ME. Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes. J Biol Chem 1985; 260(4): 2374-8.
Pöschl E, Fidler A, Schmidt B, Kallipolitou A, Schmid E, Aigner T. DNA methylation is not likely to be responsible for aggrecan down regulation in aged or osteoarthritic cartilage. Ann Rheum Dis 2005; 64(3): 477–480.
Zimmermann P, Boeuf S, Dickhut A, Boehmer S, Olek S, Richter W. Correlation of COL10A1 induction during chondrogenesis of mesenchymal stem cells with demethylation of two CpG sites in the COL10A1 promoter. Arthritis Rheum 2008; 58(9): 2743–2753.
Goldring MB, Tsuchimochi K, Ijiri K. The Control of Chondrogenesis. J Cell Biochem 2006; 97(1): 33-44.
Hardingham TE, Oldershaw RA, Tew SR. Cartilage, SOX9 and Notch signals in chondrogenesis J Anat 2006; 209(4): 469-80..
Ezura Y, Sekiya I, Koga H, Muneta T, Noda M. Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium-derived mesenchymal stem cells. Arthritis Rheum 2009; 60(5): 1416-26.
Kim KI, Park YS, Im GI. Changes in the epigenetic status of the SOX-9 promoter in human osteoarthritic cartilage. J Bone Miner Res 2013; 28(5): 1050-60.
Kevorkian L, Young DA, Darrah C, Donell ST, Shepstone L, Porter S, Brockbank SM, Edwards DR, Parker AE, Clark IM. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum 2004; 50(1): 131-41.
Roach HI, Yamada N, Cheung KS, Tilley S, Clarke NM, Oreffo RO, Kokubun S, Bronner F. Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions. Arthritis Rheum 2005; 52(10): 3110–3124
Hashimoto K, Otero M, Imagawa K, de Andrés MC, Coico JM, Roach HI, Oreffo RO, Marcu KB, Goldring MB. Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1 beta (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites. J Biol Chem 2013; 288(14): 10061-72.
Bui C, Barter MJ, Scott JL, Xu Y, Galler M, Reynard LN, Rowan AD, Young DA. cAMP response element binding (CREB) recruitment following a specific CpG demethylation leads to the elevated expression of the matrix metalloproteinase 13 in human articular chondrocytes and osteoarthritis. FASEB J. 2012; 26(7): 3000–11.
Cheung KS, Hashimoto K, Yamada N, Roach HI. Expression of ADAMTS-4 by chondrocytes in the surface zone of human osteoarthritic cartilage is regulated by epigenetic DNA de-methylation. Rheumatol Int 2009; 29(5): 525-34.
de Andrés MC, Maneiro E, Martín MA, Arenas J, Blanco FJ. Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism. Arthritis Res Ther 2013; 15(5): R115.
Taskiran D, Stefanovic-Racic M, Georgescu H, Evans C. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1. Biochem Biophys Res Commun 1994; 200(1): 142-8.
de Andres, MC, K Imagawa, K Hashimoto, et al. Loss of methylation in CpG sites in the NF-kappaB enhancer elements of iNOS is responsible for gene induction in human articular chondrocytes. Arthritis Rheum 2013; 65(3): 732-42.
Imagawa K, de Andrés MC, Hashimoto K, Pitt D, Itoi E, Goldring MB, Roach HI, Oreffo RO. The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis. Biochem Biophys Res Commun 2011; 405(3): 362-7.
Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: latest findings and interpretations. Ther Adv Musculoskelet Dis 2013; 5(2): 77-94.
Gordeladge JO, Drevon CA, Syversen U, Reseland JE. Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: impact on differentiation markers, apoptosis, and osteoclastic signaling. J Cell Biochem 2002; 85(4): 825–36.
Iliopoulos D, Malizos KN, Tsezou A. Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention. Ann Rheum Dis 2007; 66(12): 1616-21.
Hashimoto K, Oreffo RO, Gibson MB, et al. DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes. Arthritis Rheum. 2009; 60(11): 3303–13.
de Andrés MC, Imagawa K, Hashimoto K, Gonzalez A, Goldring MB, Roach HI, Oreffo RO. Suppressors of cytokine signalling (SOCS) are reduced in osteoarthritis. Biochem Biophys Res Commun 2011; 407(1): 54-9.
Chubinskaya S, Otten L, Soeder S, Borgia JA, Aigner T, Rueger DC, Loeser RF. Regulation of chondrocyte gene expression by osteogenic protein-1. Arthritis Res Ther. 2011; 13(2): R55.
Loeser RF, Im HJ, Richardson B, Lu Q, Chubinskaya S. Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage. Osteoarthritis Cartilage 2009; 17(4): 513-7.
Buxton P, Edwards C, Archer CW, Francis-West P. Growth/differentiation factor-5 (GDF-5) and skeletal development. J Bone Joint Surg Am. 2001; 83-A Suppl 1(Pt 1): S23-30.
Miyamoto Y, Mabuchi A, Shi D, Kubo T, Takatori Y, Saito S, Fujioka M, Sudo A, Uchida A, Yamamoto S, Ozaki K, Takigawa M, Tanaka T, Nakamura Y, Jiang Q, Ikegawa S. A functional polymorphism in the 5' UTR of GDF5 is associated with susceptibility to osteoarthritis. Nat Genet 2007; 39(4): 529-33.
Chapman K, Takahashi A, Meulenbelt I, Watson C, Rodriguez-Lopez J, Egli R, Tsezou A, Malizos KN, Kloppenburg M, Shi D, Southam L, van der Breggen R, Donn R, Qin J, Doherty M, Slagboom PE, Wallis G, Kamatani N, Jiang Q, Gonzalez A, Loughlin J, Ikegawa S. A meta-analysis of European and Asian cohorts reveals a global role of a functional SNP in the 5' UTR of GDF5 with osteoarthritis susceptibility Hum Mol Genet 2008; 17(10): 1497-504.
Egli RJ, Southam L, Wilkins JM, Lorenzen I, Pombo-Suarez M, Gonzalez A, Carr A, Chapman K, Loughlin J. Functional analysis of the osteoarthritis susceptibility-associated GDF5 regulatory polymorphism. Arthritis Rheum 2009; 60(7): 2055-64.
Reynard LN, Bui C, Canty-Laird EG, Young DA, Loughlin J. Expression of the osteoarthritis-associated gene GDF5 is modulated epigenetically by DNA methylation. Hum Mol Genet 2011; 20(17): 3450-60.
Delgado-Calle J, Fernandez AF, Sainz J, Zarrabeitia MT, Sañudo C, García-Renedo R, Pérez-Núñez MI, García-Ibarbia C, Fraga MF, Riancho JA.. Genome-wide profiling of bone reveals differentially methylated regions in osteoporosis and osteoarthritis. Arthritis Rheum. 2013; 65(1): 197–205.
Furumatsu T, Tsuda M, Yoshida K, Taniguchi N, Ito T, Hashimoto M, Ito T, Asahara H. Sox9 and p300 cooperatively regulate chromatin-mediated transcription. J Biol Chem 2005; 280(42): 35203-8.
Young DA, Lakey RL, Pennington CJ, Jones D, Kevorkian L, Edwards DR, Cawston TE, Clark IM. Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption. Arthritis Res Ther. 2005; 7(3): R503-12.
Wang X, Song Y, Jacobi JL, Tuan RS. Inhibition of histone deacetylases antagonized FGF2 and IL-1β eta effects on MMP expression in human articular chondrocytes. Growth Factors 2009; 27(1): 40-9.
Chabane N, Zayed N, Afif H, Mfuna-Endam L, Benderdour M, Boileau C, Martel-Pelletier J, Pelletier JP, Duval N, Fahmi H. Histone deacetylase inhibitors suppress interleukin-1beta-induced nitric oxide and prostaglandin E2 production in human chondrocytes. Osteoarthritis Cartilage. 2008 Oct; 16(10): 1267-74.
Vega RB, Matsuda K, Oh J, Barbosa AC, Yang X, Meadows E, McAnally J, Pomajzl C, Shelton JM, Richardson JA, Karsenty G, Olson EN. Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell 2004; 119(4): 555-66.
Hong S, Derfoul A, Pereira-Mouries L, Hall DJ. A novel domain in histone deacetylase 1 and 2 mediates repression of cartilage-specific genes in human chondrocytes. FASEB J. 2009; 23(10): 3539-52.
Higashiyama R, Miyaki S, Yamashita S, Yoshitaka T, Lindman G, Ito Y, Sasho T, Takahashi K, Lotz M, Asahara H. Correlation between MMP-13 and HDAC7 expression in human knee osteoarthritis. Mod Rheumatol 2010; 20(1): 11-7
Huh YH, Ryu JH, Chun JS. Regulation of type II collagen expression by histone deacetylase in articular chondrocytes. J Biol Chem 2007; 282(23): 17123-31.
Saito T, Nishida K, Furumatsu T, Yoshida A, Ozawa M, Ozaki T. Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes. Osteoarthritis Cartilage 2013; 21(1): 165-74.
Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997; 89: 755-64.
Kamekura S, Kawasaki Y, Hoshi K, Shimoaka T, Chikuda H, Maruyama Z, Komori T, Sato S, Takeda S, Karsenty G, Nakamura K, Chung UI, Kawaguchi H.. Contribution of runt-related transcription factor 2 to the pathogenesis of osteoarthritis in mice after induction of knee joint instability. Arthritis Rheum 2006; 54(8): 2462-70
Franceschi RT, Xiao G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem 2003; 88(3): 446-54.
Chen WP, Bao JP, Hu PF, Feng J, Wu LD. Alleviation of osteoarthritis by trichostatin A, a histone deacetylase inhibitor, in experimental osteoarthritis. Mol Biol Rep 2010; 37(8): 3967-72.
El Mansouri FE, Chabane N, Zayed N, Kapoor M, Benderdour M, Martel-Pelletier J, Pelletier JP, Duval N, Fahmi H.l. Contribution of H3K4 methylation by SET-1A to interleukin-1-induced cyclooxygenase 2 and inducible nitric oxide synthase expression in human osteoarthritis chondrocytes. Arthritis Rheum 2011; 63(1): 168e79.
Rodova, M. Lu Q, Li Y, Woodbury BG, Crist JD, Gardner BM, Yost JG, Zhong XB, Anderson HC, Wang J. Nfat1 regulates adult articular chondrocyte function through its age-dependent expression mediated by epigenetic histone methylation. J Bone Miner Res. 2011; 26(8): 1974-86.
Michan S, Sinclair D. Sirtuins in mammals: insights into their biological function. Biochem J 2007; 404(1): 1-13.
Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol 2010; 5: 253-95.
Dvir-Ginzberg M, Gagarina V, Lee EJ, Hall DJ. Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase. J Biol Chem. 2008; 283(52): 36300-10.
Takayama K, Ishida K, Matsushita T, Fujita N, Hayashi S, Sasaki K, Tei K, Kubo S, Matsumoto T, Fujioka H, Kurosaka M, Kuroda R. SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum 2009; 60(9): 2731-40.
Gagarina V, Gabay O, Dvir-Ginzberg M, Lee EJ, Brady JK, Quon MJ, Hall DJ. SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway. Arthritis Rheum 2010; 62(5): 1383-92.
Fujita N, Matsushita T, Ishida K, Kubo S, Matsumoto T, Takayama K, Kurosaka M, Kuroda R. Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions. J Orthop Res 2011; 29(4): 511-5.
Dvir-Ginzberg M, Gagarina V, Lee EJ, Booth R, Gabay O, Hall DJ. Tumor necrosis factor a-mediated cleavage and inactivation of sirT1 in human osteoarthritic chondrocytes. Arthritis Rheum 2011; 63(8): 2363-73.
Gabay O, Oppenheimer H, Meir H, Zaal K, Sanchez C, Dvir-Ginzberg M. Increased apoptotic chondrocytes in articular cartilage from adult heterozygous SirT1 mice. Ann Rheum Dis. 2012; 71(4): 613–6.
Matsushita T, Sasaki H, Takayama K, Ishida K, Matsumoto T, Kubo S, Matsuzaki T, Nishida K, Kurosaka M, Kuroda R. The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes. J Orthop Res 2013; 31(4): 531-7.
Moon MH, Jeong JK, Lee YJ, Seol JW, Jackson CJ, Park SY. SIRT1, a class III histone deacetylase, regulates TNF-α-induced inflammation in human chondrocytes. Osteoarthritis Cartilage 2013; 21(3): 470-80.
Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, Mills AA, Elledge SJ, Anderson KV, Hannon GJ. Dicer is essential for mouse development. Nat Genet 2003; 35(3): 215-7.
Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A 2005; 102(31): 10898-903.
Kobayashi T, Lu J, Cobb BS, Rodda SJ, McMahon AP, Schipani E, Merkenschlager M, Kronenberg HM. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci U S A. 2008; 105(6): 1949-54.
Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S, Plasterk RH. MicroRNA expression in zebrafish embryonic development. Science 2005; 309(5732): 310-1.
Tuddenham L, Wheeler G, Ntounia-Fousara S, Waters J, Hajihosseini MK, Clark I, Dalmay T. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett 2006; 580(17): 4214-7.
Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F, Nakasa T, Yamashita S, Takada S, Lotz MK, Ueno-Kudo H, Asahara H. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev 2010: 24(11): 1173–1185
Nakamura Y, Inloes JB, Katagiri T, Kobayashi T. Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets dnpep to modulate bone morphogenetic protein signaling. Mol Cell Biol 2011; 31(14): 3019-28.
Nakamura Y, He X, Kobayashi T, Yan YL, Postlethwait JH, Warman ML. Unique roles of microRNA140 and its host gene WWP2 in cartilage biology. J Musculoskelet Neuronal Interact. 2008; 8: 321–2.
Yang J, Qin S, Yi C, Ma G, Zhu H, Zhou W, Xiong Y, Zhu X, Wang Y, He L, Guo X. MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. FEBS Lett 2011; 585(19): 2992–7.
Miyaki S, Nakasa T, Otsuki S, Grogan SP, Higashiyama R, Inoue A, Kato Y, Sato T, Lotz MK, Asahara H. MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum 2009; 60(9): 2723–2730
Nicolas FE, Pais H, Schwach F, Lindow M, Kauppinen S, Moulton V, Dalmay T. Experimental identification of microRNA- 140 targets by silencing and overexpressing miR-140. RNA 2008; 14(12): 2513-20.
Pais H, Nicolas FE, Soond SM, Swingler TE, Clark IM, Chantry A, Moulton V, Dalmay T. Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level. RNA 2010; 16(3): 489-94.
Swingler TE, Wheeler G, Carmont V, Elliott HR, Barter MJ, Abu-Elmagd M, Donell ST, Boot-Handford RP, Hajihosseini MK, Münsterberg A, Dalmay T, Young DA, Clark IM. The expression and function of microRNAs in chondrogenesis and osteoarthritis. Arthritis Rheum 2012; 64(6): 1909-19.
Suomi S, Taipaleenmäki H, Seppänen A, Ripatti T, Väänänen K, Hentunen T, Säämänen AM, Laitala-Leinonen T. MicroRNAs regulate osteogenesis and chondrogenesis of mouse bone marrow stromal cells. Gene Regul Syst Bio 2008; 2: 177-91.
Yang B, Guo H, Zhang Y, Dong S, Ying D. The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation. BMB Rep 2011; 44(1): 28–33.
Yang B, Guo H, Zhang Y, Chen L, Ying D, Dong S. microRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One 2011; 6(7): e21679.
Martinez-Sanchez A, Dudek KA, Murphy CL. Regulation of human chondrocyte function through direct inhibition of cartilage master-regulator SOX9 by miRNA-145. J Biol Chem 2012; 287(2): 916-24.
Dudek KA, Lafont JE, Martinez-Sanchez A, Murphy CL. Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes. J Biol Chem 2010; 285(32): 24381-7.
Lin E, Kong L, Bai XH, Luan Y, Liu CJ. miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smad1. J Biol Chem 2009; 284(17): 11326–35.
Lin L, Shen Q, Zhang C, Chen L, Yu C. Assessment of the profiling microRNA expression of differentiated and dedifferentiated human adult articular chondrocytes. J. Orthop. Res 2011; 29(10): 1578–1584
Karlsen TA, Shahdadfar A, Brinchmann JE. Human primary articular chondrocytes, chondroblasts-like cells, and dedifferentiated chondrocytes: differences in gene, microRNA, and protein expression and phenotype. Tissue Eng. Part C: Methods 2011; 17(2): 219–227.
Xu J, Kang Y, Liao WM, Yu L. MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS One 2012; 7(3): e31861.
Zhong N, Sun J, Min Z, Zhao W, Zhang R, Wang W, Tian J, Tian L, Ma J, Li D, Han Y, Lu S.. MicroRNA-337 is associated with chondrogenesis through regulating TGFBR2 expression. Osteoarthritis Cartilage 2012; 20(6): 593–602.
Kim D, Song J, Jin EJ. MicroRNA-221 regulates chondrogenic differentiation through promoting proteasomal degradation of Slug by targeting Mdm2. J Biol Chem 2010; 285(35): 26900–7.
Kim D, Song J, Kim S, Chun CH, Jin EJ. MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5. Biochem Biophys Res Commun 2011; 415(4): 551-7.
Kim, D. J Song, S Kim, Park HM, Chun CH, Sonn J, Jin EJ. MicroRNA-34a modulates cytoskeletal dynamics through regulating RhoA/Rac1 crosstalk in chondroblasts. J Biol Chem 2012; 287(15): 12501-9.
Song J, Lee M, Kim D, Han J, Chun CH, Jin EJ. MicroRNA-181b regulates articular chondrocytes differentiation and cartilage integrity. Biochem Biophys Res Commun. 2013; 431(2): 210-4.
Iliopoulos D, Malizos KN, Oikonomou P, Tsezou A. Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS One. 2008; 3(11): e3740.
Jones SW, Watkins G, Le Good N, Roberts S, Murphy CL, Brockbank SM, Needham MR, Read SJ, Newham P. The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13. Osteoarthritis Cartilage. 2009; 17(4): 464-72.
Díaz-Prado S, Cicione C, Muiños-López E, Hermida-Gómez T, Oreiro N, Fernández-López C, Blanco FJ. Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes. BMC Musculoskelet Disord. 2012; 13: 144.
Murata K, Yoshitomi H, Tanida S, Ishikawa M, Nishitani K, Ito H, Nakamura T. Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis. Arthritis Res Ther. 2010; 12(3): R86.
Borgonio Cuadra VM, González-Huerta NC, Romero-Córdoba S, Hidalgo-Miranda A, Miranda-Duarte A. Altered expression of circulating microRNA in plasma of patients with primary osteoarthritis and in silico analysis of their pathways. PLoS One. 2014; 9(6): e97690.
Tardif G, Hum D, Pelletier JP, Duval N, Martel-Pelletier J. Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet. Disord 2009; 10: 148.
Liang ZJ, Zhuang H, Wang GX, Li Z, Zhang HT, Yu TQ, Zhang BD. MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1β-stimulated human articular chondrocyte C28/I2 cells. Inflamm Res 2012; 61(5): 503-9.
Akhtar N, Rasheed Z, Ramamurthy S, Anbazhagan AN, Voss FR, Haqqi TM. MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes. Arthritis Rheum 2010; 62(5): 1361-71.
Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N, Bentwich Z, Oren M. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell. 2007; 26(5): 731-43.
Abouheif MM, Nakasa T, Shibuya H, Niimoto T, Kongcharoensombat W, Ochi M. Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology (Oxford) 2010; 49(11): 2054-60.
Nakasa T, Miyaki S, Okubo A, Hashimoto M, Nishida K, Ochi M, Asahara H.. Expression of microRNA-146 in rheumatoid arthritis synovial tissue. Arthritis Rheum 2008; 58(5): 1284–92
Yamasaki K, Nakasa T, Miyaki S, Ishikawa M, Deie M, Adachi N, Yasunaga Y, Asahara H, Ochi M. Expression of MicroRNA-146a in osteoarthritis cartilage. Arthritis Rheum. 2009; 60(4): 1035-41.
Li J, Huang J, Dai L, Yu D, Chen Q, Zhang X, Dai K. miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4. Arthritis Res Ther 2012; 14(2): R75.
Li X, Gibson G, Kim JS, Kroin J, Xu S, van Wijnen AJ, Im HJ. MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis. Gene 2011; 480(1-2): 34-41.
Guan YJ, Yang X, Wei L, Chen Q. MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4. FASEB J 2011; 25(12): 4457e66.
Song J, Kim D, Lee CH, Lee MS, Chun CH, Jin EJ. MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis ofosteoarthritis. J Biomed Sci. 2013;20:31.