Gene Expression In Human Chondrocytes Research Articles

Response eQTLs, chromatin accessibility, and 3D chromatin structure in chondrocytes provide mechanistic insight into osteoarthritis risk.

∘ Comprehensive analysis of sex- and age-related global gene expression in human chondrocytes revealed differences that correlate with osteoarthritis ∘ First response eQTLs in chondrocytes treated with an OA-related stimulus ∘ Deeply sequenced Hi-C in resting and activated chondrocytes helps connect OA risk variants to their putative causal genes ∘ Colocalization analysis reveals 13 (including 10 novel) putative OA risk genes.

Inhibitory Effect of a Tankyrase Inhibitor on Mechanical Stress-Induced Protease Expression in Human Articular Chondrocytes.

We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and β-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1β in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1β, as well as the nuclear translocation of NF-κB and β-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of β-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptor-interacting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage-degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.

CITED2 mediates the cross-talk between mechanical loading and IL-4 to promote chondroprotection.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

MiR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes. At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene. The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression. Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

Corrigendum to: Effects of Arthrocen, an avocado/soy unsaponifiables agent, on inflammatory mediators and gene expression in human chondrocytes.

[This corrects the article DOI: 10.1002/2211-5463.12176.].

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

EZH2 inhibition reduces IL-1β-mediated inflammation and increases anabolic gene expression in human chondrocytes

MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism.Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured.Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3′-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects.Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

Effects of Arthrocen, an avocado/soy unsaponifiables agent, on inflammatory mediators and gene expression in human chondrocytes.

Osteoarthritis (OA) is a chronic joint disease characterized by pain and stiffness. Recently, there has been great interest in the use of plant‐derived compounds and supplements in managing the symptoms of OA. Arthrocen is a plant‐based supplement consisting of avocado and soy unsaponifiable extracts in a 1 : 2 ratio. In an effort to unravel the potential mechanisms of its action on the cellular level, we utilized an in vitro assay to study its effects on cultured human chondrocytes. By pairing this assay with protein arrays on inflammatory markers, RNA‐Seq with downstream pathway analysis, and lipidomics on eicosanoids, we were able to further define its action at the molecular level. Specifically, we found a role for Arthrocen in attenuating the inflammatory response both at the protein and mRNA level. Furthermore, we discovered that Arthrocen diminished prostaglandin E2 (PGE2) levels in response to an inflammatory trigger. Additionally, unlike traditional COX‐2 inhibitors, this response rather specifically attenuated PGE2 levels in the presence of inflammation and without lowering levels of other eicosanoids. This implies that Arthrocen could potentially bring about the reduced pain produced by COX‐2 inhibitors without the known side effects of COX‐2 inhibition.

Dose-dependent effects of adiponectin on ADAMTS-9 gene expression in human chondrocytes.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), comprising of 19 members is a family of peptidases. They have several vital functions in physiological and pathological processes in organisms. ADAMTS-9 has aggrecanolytic activity and is responsible for degradation of aggrecan mainly in articular cartilage. It is known that adiponectin is the most abundantly secreted adipokine (adipocytokines), and the characteristics of adiponectin have not been elucidated yet. It was assumed that adiponectin has anti-inflammatory effect before. However, an inflammatory feature of adiponectin was shown in researches. In our study, the effect of adiponectin on ADAMTS-9 gene expression in primary human chondrocytes was investigated. Primary human chondrocytes were exposed to adiponectin at 1, 4, 8 and 12 µg/ml doses for certain time period. Total RNA was isolated and reverse-transcribed by random primer after incubation. ADAMTS-9 and β-actin genes expression levels were determined using real-time polymerase chain reaction (qRT-PCR). The highest upregulation of ADAMTS-9 gene expression level was found at 12 µg/ml dose of adiponectin and 48 h incubation. Adiponectin is the key element in the maintenance of cartilage homeostasis. Similarly, the involvement of adiponectin in articular inflammatory diseases was demonstrated in detail. These findings bring adiponectin into central place in the research to develop adiponectin based new therapy methods for arthritic diseases. Together with these findings, our results suggest that adiponectin may be involved in the degradation of articular cartilage by increasing ADAMTS-9 gene expression (Tab. 1, Fig. 3, Ref. 35).

MicroRNA-92a-3p regulates the expression of cartilage-specific genes by directly targeting histone deacetylase 2 in chondrogenesis and degradation.

Increased activity of histone deacetylase 2 (HDAC2) has been found in patients with osteoarthritis (OA) and cartilage matrix degradation and has been shown to mediate the repression of cartilage-specific gene expression in human chondrocytes. We aimed to determine whether microRNA-92a-3p (miR-92a-3p) regulates cartilage-specific gene expression via targeted HDAC2 in chondrogenesis and degradation. miR-92a-3p expression was assessed invitro in a human mesenchymal stem cells (hMSCs) model of chondrogenesis and in normal and OA primary human chondrocytes (PHCs), and in normal and OA human cartilage by in situ hybridization. hMSCs and PHCs were transfected with miR-92a-3p or its antisense inhibitor (anti-miR-92a-3p), respectively. PHCs were transfected with miR-92a-3p or anti-miR-92a-3p for 24h before chromatin immunoprecipitation (ChIP) assay was performed with anti-ac-H3 antibody. Direct interaction between miR-92a-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of HDAC2 mRNA was confirmed by luciferase reporter assay. miR-92a-3p expression was elevated in chondrogenic and hypertrophic hMSC, while reduced in OA cartilage compared with normal cartilage. The overexpression of miR-92a-3p suppressed the activity of a reporter construct containing the 3'-UTR and inhibited HDAC2 expression in both hMSCs and PHCs, while treatment with anti-miR-92a-3p enhanced HDAC2 expression. ChIP assays showed that miR-92a-3p enhances H3 acetylation on aggrecan (ACAN), cartilage oligomeric protein (COMP) and Col2a1 promoter, and also promotes relative cartilage matrix expression. Our results suggest that miR-92a-3p regulates cartilage development and homeostasis, which directly targets HDAC2, indicating histone hyperacetylation plays an important role in increased expression of cartilage matrix.

Searching potential transcription factors regulating MMP13 overexpression via promoter CpG methylation status in human osteoarthritic chondrocytes: A microarray screening

Purpose: Matrix metalloproteinase 13 (MMP-13) is known to play important roles in the progress of osteoarthritis (OA) in human articular cartilage. Our previous studies have demonstrated that the elevated MMP13 gene expression in human chondrocytes is regulated by the CpG methylation status in its proximal promoter. Also, hypoxia-inducible factor (HIF) -2α, a transcription factor (TF) mainly induced under the hypoxic condition, was found to regulate MMP13 expression via methylation status of a specific CpG site in the proximal promoter. In this study, we screened TFs that have a potential to regulate MMP13 expression via its promoter CpG methylation status by a microarray analysis comparing healthy and OA human chondrocytes. Methods: Four healthy and five OA cartilage pieces were harvested from the human femoral head collected from the hemiarthroplasty for fracture of neck of femur (#NOF) and total hip arthroplasty for hip OA, respectively. Then mRNA was extracted (QIAGEN, RNeasy Lipid Kit®), from those cartilage pieces. The mRNA samples with RNA integrity number (RIN) >6.5 by Bioanalyzer® (Agilent Technologies) were proceeded to a microarray analysis (TORAY, 3D-Gene®). The expression level of MMP13 and various TF genes were compared between healthy and OA human chondrocytes. The TF gene expression with 2-time difference between healthy and OA chondrocytes were selected for further analysis. The potential binding sites of the selected TFs, and the presence of CpG sites in those binding sites, were detected in up to −5000bp of MMP13 promoter using TF binding site databases. Results: The relative expression levels of MMP13 were 32 ± 43 and 538 ± 337 in healthy and OA chondrocytes, respectively (P=0.022). In the microarray analysis, 41 TFs were more expressed, and 40 TFs were less expressed in OA compared to healthy chondrocytes, respectively. Of them, 13 TFs were found to be potentially bindable to up to −5000-bp of MMP13 promoter. Moreover, transcription factors FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 and E2F2 were found to have their consensus sequences close to CpG sites in the MMP13 promoter sequence. Conclusions: In addition to HIF-2α, the function of FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 or E2F2 can be also regulated by the methylation status of CpG sites in MMP13 promoter. Further investigation is needed to prove the detailed function of those transcription factors, especially for a relationship with CpG methylation status of the MMP13 promoter.

RNA binding proteins regulate anabolic and catabolic gene expression in chondrocytes

SummaryObjectiveRegulation of anabolic and catabolic factors is considered essential in maintaining the homoeostasis of healthy articular cartilage. In this study we investigated the influence of RNA binding proteins (RNABPs) in this process.DesignUsing small interfering RNA (siRNA), RNABP expression was knocked down in SW1353 chondrosarcoma cells and human articular chondrocytes. Gene expression and messenger RNA (mRNA) decay of anabolic (SOX9, Aggrecan) and catabolic (matrix metalloproteinase (MMP)13) factors were analysed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA-electromobility shift assays (EMSAs) were used to investigate RNABP interactions with the SOX9 mRNA 3′ untranslated region (UTR). Immunohistochemical localisation of MMP13 and the RNABP human antigen R (HuR) was performed in E13.5 and E16.5 mouse embryo sections.ResultsSOX9 mRNA, mRNA half-life and protein expression were increased with siRNA targeting the RNABP tristetraprolin (TTP) in both HACs and SW1353s. TTP knockdown also stimulated aggrecan mRNA expression but did not affect its stability. RNA-EMSAs demonstrated that adenine uracil (AU)-rich elements in the SOX9 mRNA 3′UTR interacted with chondrocyte proteins with three specific elements interacting with TTP. HuR knockdown significantly increased MMP13 expression and also regulated the expression of a number of known transcriptional repressors of MMP13. HuR was ubiquitously expressed within mouse embryos yet displayed regional down-regulation within developing skeletal structures.ConclusionThis study demonstrates for the first time how RNABPs are able to affect the balance of anabolic and catabolic gene expression in human chondrocytes. The post-transcriptional mechanisms controlled by RNABPs present novel avenues of regulation and potential points of intervention for controlling the expression of SOX9 and MMP13 in chondrocytes.

Differential gene expression of human chondrocytes cultured under short-term altered gravity conditions during parabolic flight maneuvers

BackgroundChondrocytes are the main cellular component of articular cartilage. In healthy tissue, they are embedded in a strong but elastic extracelluar matrix providing resistance against mechanical forces and friction for the joints. Osteoarthritic cartilage, however, disrupted by heavy strain, has only very limited potential to heal. One future possibility to replace damaged cartilage might be the scaffold-free growth of chondrocytes in microgravity to form 3D aggregates.ResultsTo prepare for this, we have conducted experiments during the 20th DLR parabolic flight campaign, where we fixed the cells after the first (1P) and the 31st parabola (31P). Furthermore, we subjected chondrocytes to isolated vibration and hypergravity conditions. Microarray and quantitative real time PCR analyses revealed that hypergravity regulated genes connected to cartilage integrity (BMP4, MMP3, MMP10, EDN1, WNT5A, BIRC3). Vibration was clearly detrimental to cartilage (upregulated inflammatory IL6 and IL8, downregulated growth factors EGF, VEGF, FGF17). The viability of the cells was not affected by the parabolic flight, but showed a significantly increased expression of anti-apoptotic genes after 31 parabolas. The IL-6 release of chondrocytes cultured under conditions of vibration was not changed, but hypergravity (1.8 g) induced a clear elevation of IL-6 protein in the supernatant compared with corresponding control samples.ConclusionTaken together, this study provided new insights into the growth behavior of chondrocytes under short-term microgravity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0095-9) contains supplementary material, which is available to authorized users.

Effect of adiponectin on a disintegrin and metalloproteinase with thrombospondin motifs-9 gene expression in human chondrocytes (CCR5P.257)

Abstract A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a family of peptidases. They comprise 19 members, and have several vital functions in organism. ADAMTS-9 with aggrecanolytic activity is responsible for the degradation of the articular cartilage components like aggrecan. Adiponectin is the most abundantly secreted adipokines (adipocytokines), and the characteristic of adiponectin is controversial. It was assumed that adiponectin has anti-inflammatory effect. However, recent studies showed the inflammatory features of adiponectin. It was aimed to evaluate the effect of adiponectin on ADAMTS-9 gene expression in human chondrocytes. Human articular chondrocytes were cultured and exposed by adiponectin at 1, 4, 8 and 12 µg/ml doses. At the end of the incubation, total RNA was reverse-transcribed by random primer. The expression levels of the ADAMTS-9 and β-actin genes were determined using real-time polymerase chain reaction. The ADAMTS-9 gene expression was found to increase after adiponectin incubation at 12 µg/ml dose. The interleukin-1β induced ADAMTS-9 gene up-regulation and the increased serum level of ADAMTS-9 in patients with osteoarthritis (OA) were reported before. Similarly, the putative involvement of adiponectin in OA and rheumatoid arthritis (RA) was demonstrated. Together with these findings, our results suggesting that adiponectin may involve in the degradation of aggrecan by increasing ADAMTS-9 gene expression.

Leptin increases ADAMTS9 gene expression in human chondrocytes

Leptin increases ADAMTS9 gene expression in human chondrocytes

AB0070 Wnt and wisp1 expression in the synovium induces cartilage damage by skewing of tgf-beta signaling via the canonical wnt signaling pathway

BackgroundMany osteoarthritis (OA) patients show significant synovial involvement. Recently, we found strong upregulation of canonical Wnts 2b and 16 and WISP1, a downstream protein, in the synovium in two murine...

FRI0041 Pro-inflammatory cytokines IL-1β and tnf-α regulate the genomic 5-hydroxymethylcytosine levels by modulating the expression and activity of tet-1 and idhs in human oa chondrocytes

Background5-hydroxymethyl cytosine (5-hmC) was recently discovered as a new epigenetic mark widely distributed in all types of tissues with varying degrees of abundance. 5-hmC is formed by the oxidation of...

Quadriceps muscle strength and its relationship to radiographic knee osteoarthritis

Quadriceps muscle strength and its relationship to radiographic knee osteoarthritis

NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis

IntroductionMicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.MethodsGene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.ResultsIn contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.ConclusionsThis is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.