A wide range of specific microRNAs have been shown to have either positive or negative effects on osteoblast differentiation and function, with consequent changes in postnatal bone mass. A number of specific targets have been identified. We previously used CrispR-Cas9 to make a miR-455 null mouse, characterizing a behavioral phenotype with age. The current study identifies a bone phenotype, starting in younger animals. At 3 weeks of age, the miR-455 null mice (both male and female) display increased length of both long bones and vertebrae and, while this difference diminishes across 1 year, it remains significant. Increased bone formation in vivo is mirrored by an increase in osteogenesis from bone marrow-derived stem cells in vitro. This is accompanied by a decrease in osteoclastogenesis and osteoclast function. MicroCT analyses show increased trabecular bone and less porosity/decreased separation in the miR-455 null mouse, suggesting a more dense and stronger bone at 3 weeks of age; these differences normalize by 1 year. Gain-of-function and loss-of-function datasets show that FGF18 expression is regulated by miR-455 and FGF18 was validated as a direct target of miR-455. The regulation of FGF18 by miR-455 is a likely mediator of its effect on bone. MicroRNAs are small molecules that have profound effects on the production of proteins and therefore on the function of cells and animal physiology. We made a mouse that lacks a specific microRNA, microRNA-455. This mouse has an increase in bone length from 3 weeks of age. There is an increase in bone formation by bone-forming cells (osteoblasts) and a decrease in bone loss (mediated by osteoclasts). While several genes/proteins are altered by the absence of microRNA-455, a factor called fibroblast growth factor 18 (FGF18) is increased. This is likely to be a key mediator of the impact of microRNA-455 on bone.

Objective: Determining the effect of microRNA-544a (miR-544a) in articular chondrocytes isolated from patients affected by osteoarthritis (OA) and its role in the modulation of the Wnt signalling. Methods: Articular chondrocytes were isolated from patients undergoing joint replacement because of OA. Expression levels of miR-544a were measured by PCR and by in situ hybridization. Putative targets of miR-544a were confirmed by reporter assay and by qPCR in cells stimulated with a miR-544a mimic. The effect of miR-544a on chondrocyte metabolism was monitored by qPCR for phenotypic markers, protein expression levels of aggrecan neoepitopes/MMP-13 and modulation of alcian blue content in micromass cultures, upon stimulation with a miR-544a mimic. The expression levels of MMP-13 and Aggrecan neoepitopes in response to miR-544a stimulation was also measured in co-stimulation with Xav-939 and KN93, which are respectively β-catenin and CaMKII inhibitors. Results: Our results suggest that miR-544a enhances the activation of the Wnt-signalling in the articular chondrocytes, by downregulating the expression of components of the Wnt/β-catenin destruction complex. The expression of miR-544a is higher in chondrocytes isolated from damaged areas of the articular cartilage removed from OA patients, and can be upregulated by pro-inflammatory and pro-fibrotic cytokines. miR-544a exerts a pro-catabolic effect of articular chondrocytes, which is rescued both by the inhibition of the Wnt/β-catenin and Wnt/CaMKII signalling pathways. Conclusion: our results point to miR-544a as a new, important modulator of the Wnt signalling network within the articular cartilage suggesting a key role for microRNAs in regulating how the multiple branches of the network and their interaction modulate cartilage homeostasis. ### Competing Interest Statement The authors have declared no competing interest.

The complete molecular mechanisms underlying the pathophysiology of Alzheimer's disease (AD) remain to be elucidated. Recently, microRNA-455-3p has been identified as a circulating biomarker of early AD, with increased expression in post-mortem brain tissue of AD patients. MicroRNA-455-3p also directly targets and down-regulates APP, with the overexpression of miR-455-3p suppressing its toxic effects. Here, we show that miR-455-3p expression decreases with age in the brains of wild-type mice. We generated a miR-455 null mouse utilising CRISPR-Cas9 to explore its function further. Loss of miR-455 resulted in increased weight gain, potentially indicative of metabolic disturbances. Furthermore, performance on the novel object recognition task diminished significantly in miR-455 null mice (p = 0.004), indicating deficits in recognition memory. A slight increase in anxiety was also captured on the open field test. BACE1 and TAU were identified as new direct targets for miR-455-3p, with overexpression of miR-455-3p leading to a reduction in the expression of APP, BACE1 and TAU in neuroblastoma cells. In the hippocampus of miR-455 null mice at 14 months of age, the levels of protein for APP, BACE1 and TAU were all increased. Such findings reinforce the involvement of miR-455 in AD progression and demonstrate its action on cognitive performance.

MicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFβ1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFβ1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

Purpose: Using deep sequencing to identify novel miRNAs in human osteoarthritic cartilage, a sequence annotated as miR-3085-3p in mice and rats, was identified for the first time in man. In man, miR-3085 is located within an intron of the CRTAC1 gene which is expressed in chondrocytes. The purpose of this study was to characterize this microRNA and to define its function. Methods: Ectopic overexpression of a hairpin structure of premiR-3085 followed by qRT-PCR and Ago pulldown were used to prove that miR-3085 is a miRNA. Expression of miR-3085 and CRTAC1 was investigated by qRT-PCR. Expression was analysed in micromass or monolayer culture of primary human articular chondrocytes stimulated with TGFβ1, IL-1β or Wnt3A; or after manipulation of SOX9, p50, p65. Functional interaction between the miRNA and Smad, NFκB and Wnt signalling pathways was explored using luciferase reporters and confirmed by Western blot. Novel targets of miR-3085-3p were characterized by gene expression profiles in human chondrocytes transiently transfected with miRNA mimic or inhibitor, confirmed by qRT-PCR. Direct targets were confirmed by cloning the 3’UTR downstream of a luciferase reporter. Results: Expression of the mature miR-3085-3p was strongly increased when a hairpin structure consisting of mature miR-3085 strands together with the flanking regions was overexpressed. Ago-pulldown showed that miR-3085 was an active microRNA. TGFβ1 and IL-1β increased, whilst Wnt3a had a little effect on, expression of miR-3085-3p and CRTAC1 in primary human articular chondrocytes. IL-1β-induced miR-3085-3p and CRTAC1 expression partly through NFκB. Ectopic expression of SOX9 increased the expression of CRTAC1 but not the miR-3085-3p itself. Functionally, miR-3085-3p acts as a negative regulator of TGFβ1/Smad, IL-1β/NFκB and Wnt3a/β-catenin signalling pathways. The IL-1-induced expression of MMP13 and the TGFβ1-induced expression of TIMP3 were both repressed by miR-3085. Western blot to look at expression, activation and nuclear localization of p65, Smad2, Smad3, Smad4, and β-catenin showed that overexpression of miR-3085-3p lead to a decrease in p65, p-p65, Smad2, Smad3, p-Smad3, Smad4, β-catenin, p-β-catenin in either whole cell lysate or a nuclear fraction. A number of genes were identified as direct targets of miR-3085-3p: COL2A1, ACAN, SMAD2, SMAD3, SMAD4, MyD88, PODXI001, KLF6, Ly6E, DDITL, TMEM140, MXD1, Scinp1, TSCD0003, APOL3, CD248. Conclusions: These data experimentally prove miR-3085 as a miRNA. It is regulated by a number of factors known to have a role in cartilage homeostasis and it has functional impact on relevant signalling pathways. We have identified a number of novel genes which are direct targets of miR-3085. Future work will continue to ascertain the pathways via which miR-3085 can regulate the development and progression of OA.

MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family was also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFβ1 decreased expression of miR-29a, b, and c (3p) in primary chondrocytes, whilst IL-1β increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB, and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, and CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways.

Small non-coding RNAs known as microRNAs (miRs) have recently been recognised as important regulators of gene expression in human cells. Whilst originally mooted to fine-tune gene expression, it is now clear that they can have profound effects on physiology and disease. A role for microRNAs in chondrogenesis and osteoarthritis has become clear over the last decade. Deletion of the machinery for microRNA biogenesis specifically in the cartilage of mice has shown that, as a class, microRNAs influence the development of the skeleton and homeostasis of articular cartilage. Identifying roles for individual microRNAs has been more difficult, with the main in vivo data coming from miR-140. This session will review the role of microRNAs in the molecular pathogenesis of osteoarthritis (OA). It will present data on the regulation of microRNAs by relevant factors and their impact on intracellular signalling. Key targets of microRNAs in OA will also be explored. It will touch upon the use of microRNAs as circulating biomarkers of disease and the potential for microRNA-based therapy. The focus of the Clark lab has been on the role and function of microRNAs in cartilage, particularly miR-455, the miR-29 family, miR-140 and miR-3085. MicroRNA-455 is genomically located within an intron of COL27A1. Collagen XXVII is expressed in cartilage, suggesting function of the miR in this tissue. We initially described a role for miR-455 in TGFbeta signalling, but have more recently uncovered function in Wnt signalling and in the regulation of Sirt1. The miR-29 family have been well-researched and are known e.g. to regulate collagen gene expression, giving them a role in fibroses. We identified miR-29b as one of only two miRs which was regulated at an early time point after surgery in the murine DMM (“destabilisation of the medial meniscus”) model of OA. Potential targets of miR-29 were regulated in the opposite direction to the miR, suggesting function. The miR-29 family were regulated in many models of chondrocyte differentiation and in human end-stage OA. In chondrocytes we have shown that miR-29 is negatively regulated by Sox9 and negatively regulates a number of key intracellular signalling pathways in OA. We have identified novel direct targets in the Wnt pathway and we have also shown that miR-29 directly targets a number of ADAMTS protease genes. We used RNA-Seq to explore the full range of miRs expressed by human articular chondrocytes from OA patients. This showed that the so-called passenger strand of miR-140, miR-140-3p was more highly expressed than the guide strand, miR-140-5p in newly isolated osteoarthritic chondrocytes. We have now shown that miR-140-3p directly targets a number of enzymes in the heparan sulphate proteoglycan synthesis pathway. These studies also identified miR-3085 in human chondrocytes. This miR had only previously been annotated in rodents where it was presumed intergenic. However, in man it is located in the final intron of the CRTAC1 gene, which codes for cartilage acidic protein 1. We have shown it to directly target ITGA5, the integrin alpha5 gene, but it also strongly induces interleukin-1 signalling in chondrocytes. Disclosure of Interest None declared

Purpose: MicroRNAs are short endogenous non-coding RNA molecules, typically 19-25 nucleotides in length, which negatively regulate gene expression. In osteoarthritis (OA), several genes necessary for cartilage homeostasis are aberrantly expressed, with a number of microRNAs implicated in this process. However, our knowledge of the earliest stages of OA, prior to the onset of irreversible changes, remains limited. The purpose of this study was to identify microRNAs involved across the time-course of OA using both a murine model and human cartilage, and to define their function. Methods: Total RNA was purified from whole knee joints taken from mice when underwent destabilisation of the medial meniscus (DMM) surgery at day 1, 3 and 7 post-surgery. Expression of microRNAs (Exiqon) and mRNAs (Illumina) was performed using microarray and further validated by qRT-PCR. Change in expression of the miR-29 family (miR-29a, b, c) was analysed by qRT-PCR in SW1353 cell lines and primary human chondrocytes stimulated with TGFβ1, TGFβ3, IL-1β and Wnt3A; or after overexpression (plasmid) or knockdown (siRNA) of SOX9. miR-29a/b1 promoter activity was measured using promoter-reporter constructs. The functional interaction between miR-29 and TGFβ, NFκB and canonical Wnt signalling pathways were explored using luciferase reporters. Novel targets of the miR-29 family were characterized by gene expression profiles in human chondrocytes transiently transfected with miRNA mimic or inhibitor, confirmed by qRT-PCR. Direct targets were confirmed by cloning the 3’UTR downstream of a luciferase reporter. Results: MicroRNA expression in whole mouse joints post DMM surgery increased over 7 days. At day 1 and 3, the expression of only 4 microRNAs altered significantly. At day 7, 19 microRNAs were upregulated and 15 downregulated. Among the modulated miRNAs, the expression of miR-29b was regulated in the opposite direction to its potential targets. Interestingly, in end-stage human OA cartilage, the miR-29 family were also regulated compared to control cartilage. Both TGFβ1 and TGFβ3 decreased expression of microRNA29a, b and c whilst IL-1β increased their expression. Functionally, the microRNA 29 family were all able to negatively regulate the Smad, NFκB and canonical Wnt signalling pathways. SOX9 directly represses the expression of microRNA 29a and b via binding to their promoter. Gene expression profiles of gain-and-loss-of-function revealed regulation of a large number of previously recognised extracellular matrix-associated genes as well as an additional subset of protease and Wnt signalling pathway-related genes. Among these genes, ADAMTS5, ADAMTS6, ADAMTS14, ADAMTS17, ADAMTS19, FZD3, DVL3, FRAT2, CK2A2 were experimentally validated as direct targets of the miR-29 family. Conclusions: These data identify the microRNA 29 family as microRNAs which act early in the development of OA. We have shown that they are regulated by a number of factors known to be important in OA and that they have functional impact on several relevant signalling pathways. We have identified a number of novel genes which are direct targets of the miR-29 family. Future work will continue to ascertain the pathways via which the miR-29 family can regulate the development and progression of OA.

Purpose: Differentiating mesenchymal stem cells are often used as an alternative supply of chondrocytes that allow observations not only into the molecular signalling events that occur during chondrogenesis, but also in mature cartilage tissue. The resulting tissue can then also be used to model various cartilage pathologies or to attempt cartilage repair. It is difficult to generate the required phenotype of the chondrocytes/tissue produced and models may therefore not accurately reflect the appropriate type of cartilage tissue required, which has different functions and properties depending on the location within the body. Since osteoarthritis is a disease that degrades articular cartilage, the purpose of this study was to identify a culturing model with physiological relevance that specifically produces articular cartilage-like tissue. Methods: ATDC5 chondrogenic precursor cells were grown in three-dimensional, high-density micromass, stimulated with a combination of insulin (which induces differentiation in ATDC5 cells) and ascorbic acid over time, and directly compared to traditional ATDC5 differentiation in monolayer by insulin stimulation alone. Quantitative (q)RT-PCR was used to identify regulation of various genes related to chondrogenesis, chondrocyte maturation/hypertrophy (reminiscent of growth plate cartilage) and markers of the articular cartilage phenotype. Confirmation of some genes was observed at the protein level by immunofluorescence and histological staining. Results: ATDC5 cells induced to differentiate in the traditional monolayer culture developed heterogeneously, forming aggregates termed "cartilage nodules", which stained for Alcian blue and increased in number with ascorbic acid stimulation. Areas between nodules did not stain with Alcian blue, indicating a heterogeneous mixture of chondrogenic and non-chondrogenic areas, confirmed by Type II Collagen immunofluorescence. In contrast, central micromasses remained as one large nodule, with noticeably extensive extra-cellular matrix deposition, staining deeply and uniformly with Alcian blue, indicating more homogenous differentiation. By qRT-PCR, differentiating micromasses stimulated with ascorbic acid for three weeks had a 231-fold increase in Col2a1 expression (Type II Collagen) compared to traditional differentiating monolayer cultures with stimulation by insulin alone. Frzb, a secreted Wnt antagonist previously described as a marker for articular cartilage, was increased 35-fold in the same comparison. Col10a1 (Type X Collagen), a hypertrophic region marker, was increased with micromass culture, but protein was immunolocalised to a small region within the central micromasses surrounded by extensive Type II Collagen staining. Other markers of hypertrophic differentiation, which would resemble a more growth plate-like phenotype, were either not expressed (Mmp13) or did not demonstrate altered expression (Runx2). Taken together, these results suggest that a marked difference in gene expression is seen when ATDC5 cells are cultured in micromass with ascorbic acid. Conclusions: Using the traditional culturing method in monolayer with insulin stimulation alone, ATDC5 chondrogenesis occurs heterogeneously, forming separate cartilage nodules, which we showed can be increased in number with ascorbic acid stimulation. Using a three-dimensional culturing technique, which is more physiologically relevant, and combining this with ascorbic acid stimulation, we showed that chondrogenesis was both more homogenous in nature and also highly enhanced expression of markers of chondrogenesis and of articular cartilage. We therefore suggest that this modified culture system for ATDC5 cells provides a good model for studying articular cartilage.

The etiology of osteoarthritis (OA) is complex, with genetic, developmental, biochemical, and biomechanical factors contributing to the disease process. Chondrocytes in articular cartilage must express appropriate genes to achieve tissue homeostasis, and this is altered in OA. One facet of the aberrant gene expression in OA is the replay of chondrocyte differentiation with the expression of genes associated with chondrocyte hypertrophy. The pattern of gene expression and the transcription factors that control chondrogenesis are known in some detail. Mechanisms that lead to altered gene expression in OA, however, are less well understood. MicroRNAs (miRNAs) are small noncoding RNAs that have recently been recognized as important regulators of gene expression in human cells. A number of miRNAs are regulated across chondrogenesis, and their function is beginning to be delineated. Similarly, miRNAs are differentially expressed in OA cartilage compared to normal tissue. MicroRNA-140 (miR-140), which is highly and selectively expressed in cartilage, has been the focus of much work to date, though the full gamut of its actions is still to be defined. Many other regulated miRNAs likely act as a network to control cartilage homeostasis, catabolism, and repair. Chondrocytes are the sole cell type in cartilage, and they produce and maintain the extracellular matrix (ECM) that gives the tissue its load-bearing function (1). Chondrocytes originate from mesenchymal stem cells (MSCs) through chondrogenic differentiation (2). Investigation of the mechanisms mediating chondrogenic differentiation of MSCs as well as regulation of their functions will contribute to a better understanding of skeletal development and new strategies for treating diseases such as OA. Chondrogenesis and chondrocyte function are highly regulated by transcription and growth factors. The SOX family members SOX9, L-SOX5, and SOX6 are necessary for chondrogenic differentiation (see, for example, ref. 3). Many family members of the bone morphogenetic protein (BMP) and transforming growth factor (TGF ) signaling pathways have also been shown to control chondrogenesis (2). An additional level of regulation mediated by miRNAs has been identified (4), and miRNAs may represent novel therapeutic targets for pharmacologic control of skeletal diseases. OA, the most prevalent degenerative joint disease, causes pain, tenderness, limitation of movement, and a variable degree of inflammation (5). OA is characterized by articular cartilage destruction due to an imbalance between the synthesis and degradation of ECM components, mainly type II collagen and the proteoglycan aggrecan. Matrix-degrading enzymes, e.g., the matrix metalloproteinases (MMPs) and ADAMTS, play important roles (1). The pathogenesis of OA is complex and poorly understood but involves the interaction of multiple factors, ranging from genetic predisposition to mechanical and environmental components (5). Studies are in progress to define molecular mechanisms underlying OA, including the roles of specific miRNAs in, e.g., phenotype shift, apoptosis, and regulation of gene expression in chondrocytes (6). MicroRNA-140, the major miRNA implicated in OA to date, plays a role in chondrogenesis and cartilage development (7–9). The knockout or overexpression of miR140 in vivo has profound effects on the development of OA (10,11). Other miRNAs appear to follow this pattern (see, for example, ref. 12), but it is likely that further miRNAs that contribute to OA play a role in, for example, mechanotransduction or inflammation. The utility of miRNAs may be in the diagnosis of OA, tissue Supported by the Vietnamese Ministry of Education and Training (Project 322 grant to Ms Le) and Arthritis Research UK (program grant 19424 to Drs. Swingler and Clark). Linh T. T. Le, MSc, Tracey E. Swingler, PhD, Ian M. Clark, PhD: University of East Anglia, Norwich, UK. Address correspondence to Ian M. Clark, PhD, Biomedical Research Centre, School of Biological Sciences, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK. E-mail: i.clark@uea.ac.uk. Submitted for publication September 11, 2012; accepted in revised form April 23, 2013.

OBJECTIVE:To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro.METHODS:A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro.RESULTS:Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression.CONCLUSION:Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFβ/Smad signalling activity linked to epithelial–mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFβ, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFβ-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFβ/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFβ-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets.

Purpose: We have previously shown that Dkk3 expression is increased in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The factors regulating Dkk3 expression in cartilage and the effect of Dkk3 on chondrocyte function are poorly ascribed. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFβ signalling in other cell systems. Methods: Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1 (IL1) and oncostatin-M (OSM). The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)- and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/-Wnt3a, TGFβ and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/-TGFβ or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFβ signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/- Dkk3 and proteoglycan output assessed using alcian blue staining. β-catenin was silenced in primary HAC prior to TGFβ and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results: Dkk3 expression was decreased in primary HAC following IL1/OSM treatment. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonized Wnt signalling, decreasing Wnt3a-induced AXIN2 expression and luciferase expression from the TOPFlash reporter. Dkk3 also antagonized Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFβ signalling, increasing TGFβ-induced TIMP3 and ADAM12 expression and TGFβ-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonized Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. β-catenin knockdown did not significantly alter TGFβ- or Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Knockdown of Dkk3 in primary HAC resulted in altered expression of a number of genes, with decreases in DICER and BMPR2, and increases in CTGF detected. Conclusions: OA pathogenesis is likely regulated by a multitude of factors relating to cell signalling including the balance of cytokines in the articular joint. Dkk3 expression is increased in OA but can be regulated IL1 and OSM. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA may alter chondrocyte phenotype through alterations in miRNA activity. The ability of Dkk3 to antagonize Wnt, enhance TGFβ and antagonize Activin signalling would have multiple effects on chondrocyte activity. These results imply that Dkk3 could influence multiple OA-relevant processes, protect cartilage from degradation and be important in cartilage development and homeostasis.

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.