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Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis. DOT1L is one of the few genes linked to osteoarthritis by human GWAS. Here the authors show that DOT1L-dependent histone methylation protects homeostasis of articular chondrocytes by SIRT1-dependent inhibition of canonical WNT signalling, and that inhibition of DOT1L can drive osteoarthritic disease in mice.

Osteoarthritis is the most common joint disease and a global leading cause of pain and disability. Current treatment is limited to symptom relief, yet there is no disease-modifying therapy. Its multifactorial etiology includes excessive activation of Wnt signaling, but how Wnt causes joint destruction remains poorly understood. Here, we identify that Wnt signaling promotes the transcription of insulin-like growth factor 1 (IGF1) in articular chondrocytes and that IGF1 is a major driver of Wnt-induced joint damage. Male mice with cartilage-specific Igf1 deficiency are protected from Wnt-triggered joint disease. Mechanistically, Wnt-induced IGF1 transcription depends on β-catenin and binding of Wnt transcription factor TCF4 to the IGF1 gene promoter. In a clinically relevant mouse model of post-traumatic osteoarthritis, cartilage-specific deletion of Igf1 protects against the disease in male mice. IGF1 silencing in chondrocytes from patients with osteoarthritis restores a healthy molecular profile. Our findings reveal that reducing Wnt-induced IGF1 is a potential therapeutic strategy for osteoarthritis. Osteoarthritis is a common, yet untreatable, joint disease associated with high Wnt signaling, but how Wnt causes joint destruction is unknown. Here, the authors show that Wnt induces IGF1 which leads to joint damage, and targeting this mechanism offers a potential therapeutic opportunity in osteoarthritis.

Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, only a few studies have investigated their potential as natural anti-inflammatory drug candidate compounds. The present study investigates the anti-inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or recombinant human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS-induced NO production without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC50 values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents.

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2's inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders.

Psoriasis is a chronic inflammatory disorder affecting skin and joints that results from immunological dysfunction such as enhanced IL‐23 induced Th‐17 differentiation. IkappaB‐Zeta (IκBζ) is an atypical transcriptional factor of the IκB protein family since, contrary to the other family members, it positively regulates NF‐κB pathway by being exclusively localized into the nucleus. IκBζ deficiency reduces visible manifestations of experimental psoriasis by diminishing expression of psoriasis‐associated genes. It is thus tempting to consider IκBζ as a potential therapeutic target for psoriasis as well as for other IL23/IL17‐mediated inflammatory diseases. In this review, we will discuss the regulation of expression of NFKBIZ and its protein IκBζ, its downstream targets, its involvement in pathogenesis of multiple disorders with emphasis on psoriasis and evidences supporting that inhibition of IκBζ may be a promising alternative to current therapeutic managements of psoriasis. Expression of NFKBIZ is stimulated by cytokines such as IL‐17, TNFα, IL‐1β and IL‐36. Increased expression of IκBζ eventually activates NF‐κB leading to increased expression of various proinflammatory cytokines, chemokines, as well as psoriasis‐associated genes. This cascade of molecules causes recruitment of neutrophils and other immune‐mediated inflammatory cells, which consequently leads to the development of psoriasis‐like skin inflammation.

BackgroundSMOC2, a secreted calcium-binding protein of the BM-40/SPARC family was identified from a chondrogenic extract of articular cartilage and is increased in osteoarthritic cartilage. Previous research demonstrated that Wnt signalling needs to be carefully balanced in cartilage to ensure homeostasis. We evaluated the effect of SMOC2 on in vitro chondrogenesis and its relationship with Wnt signalling.Materials and methodsSmoc2 was stably overexpressed or knocked down in ATDC5 cells cultured as micromasses. Impact of SMOC2 on chondrogenesis was evaluated by gene expression analysis of chondrogenic markers (Acan, Col2a1, Col10a1) and by Sirius red, Alcian Blue and Alizarin red staining to evaluate collagens, proteoglycans and mineralization. Femoral head caps of 6 weeks old mice were isolated and stimulated with IL-1β to induce cartilage breakdown. Influence of SMOC2 on cartilage breakdown was evaluated by co-stimulating femoral head caps with recombinant SMOC2 protein. Glycosaminoglycan release in the culture medium was measured by performing a DMMB assay. The influence of SMOC2 on canonical Wnt signalling was assessed by performing a TOP/FOP-flash reporter assay in ATDC5 cells. Human articular chondrocytes were stimulated with Wnt agonist CHIR99021 and recombinant SMOC2 protein to validate the effect of SMOC2 on Wnt signalling. Gene expression of canonical Wnt markers was analysed.ResultsGene expression of chondrogenic markers and staining intensity of Alcian blue and Alizarin red in ATDC5 micromasses was strongly reduced in Smoc2 overexpressing cells compared to control. Mice femoral head caps stimulated with recombinant SMOC2 protein showed an increased glycosaminoglycan release after IL-1β treatment in culture medium in comparison to IL-1β stimulation alone. ATDC5 cells stimulated with recombinant SMOC2 exhibit decreased luciferase activity in the Wnt reporter assay. In contrast we observed an increased luciferase activity in ATDC5 cells with transient Smoc2 knockdown. This inhibitory effect of Smoc2 on canonical Wnt signalling was also observed in human articular chondrocytes.ConclusionSmoc2 negatively affects chondrogenic differentiation of precursor cells and increases IL-1β induced breakdown in mice cartilage. Inhibition of canonical Wnt signalling is identified as contributing mechanism.

Osteoarthritis is the most prevalent joint disease worldwide, and it is a leading source of pain and disability. To date, this disease lacks curative treatment, as underlying molecular mechanisms remain largely unknown. The histone methyltransferase DOT1L protects against osteoarthritis, and DOT1L-mediated H3K79 methylation is reduced in human and mouse osteoarthritic joints. Thus, restoring DOT1L function seems to be critical to preserve joint health. However, DOT1L-regulating molecules and networks remain elusive, in the joint and beyond. Here, we identified transcription factors and networks that regulate DOT1L gene expression using a potentially novel bioinformatics pipeline. Thereby, we unraveled a possibly undiscovered link between the hypoxia pathway and DOT1L. We provide evidence that hypoxia enhanced DOT1L expression and H3K79 methylation via hypoxia-inducible factor-1 α (HIF1A). Importantly, we demonstrate that DOT1L contributed to the protective effects of hypoxia in articular cartilage and osteoarthritis. Intra-articular treatment with a selective hypoxia mimetic in mice after surgical induction of osteoarthritis restored DOT1L function and stalled disease progression. Collectively, our data unravel a molecular mechanism that protects against osteoarthritis with hypoxia inducing DOT1L transcription in cartilage. Local treatment with a selective hypoxia mimetic in the joint restores DOT1L function and could be an attractive therapeutic strategy for osteoarthritis.

ObjectivesSuramin is an old drug used for the treatment of African sleeping sickness. We investigated therapeutic repositioning of suramin to protect against cartilage damage, as suramin may interact with tissue inhibitor of metalloproteinase-3 (TIMP3).MethodsIn vitro extracellular matrix (ECM) accumulation and turnover in the presence or absence of suramin were studied in the ATDC5 micromass model of chondrogenesis and in pellet cultures of human articular chondrocytes from osteoarthritis and control patients, by gene expression, protein analysis, colorimetric staining, immunoprecipitation, fluorimetric analysis and immunohistochemistry. To study suramin in vivo, the drug was injected intra-articularly in the papain model of joint damage. Disease severity was analysed by histology, immunohistochemistry and contrast-enhanced nanofocus CT.ResultsIn ATDC5 micromasses, suramin increased TIMP3 levels and decreased the activity of matrix metalloproteinases (MMPs) and aggrecanases. Suramin treatment resulted in increased glycosaminoglycans. This effect on the ECM was blocked by an anti-TIMP3 antibody. Direct interaction between suramin and endogenous TIMP3 was demonstrated in immunoprecipitates. Mice treated intra-articularly with suramin injections showed reduced cartilage damage compared with controls, with increased TIMP3 and decreased MMP and aggrecanase activity. Translational validation in human chondrocytes confirmed increased TIMP3 function and reduced cartilage breakdown after suramin treatment.ConclusionSuramin prevented loss of articular cartilage in a mouse model of cartilage damage. The effects appear to be mediated by a functional increase of TIMP3 and a subsequent decrease in the activity of catabolic enzymes. Thus, suramin repositioning could be considered to prevent progressive cartilage damage and avoid evolution toward osteoarthritis.

PurposeDrug repositioning is a recent pharmaceutical strategy to discover new uses for market-approved drugs, with known safety profiles that can provide quick transition from the laboratory bench to bedside. Here, we investigate the influence of suramin, a polysulfonated compound that has been used in vitro to isolate tissue inhibitor of metalloproteinase (TIMP)−3 from the rat uterus. We therefore hypothesised that suramin may protect articular cartilage against osteoarthritis, by decreasing the catabolic action of tissue destructive enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) through increasing TIMP3 levels in the cartilage matrix.MethodsWe cultured differentiating ATDC5 mouse chondroprogenitor cell line in micromasses, and freshly-isolated human articular chondrocytes (hAC) in pellets, each at 200.000 cells/10 µl, in the presence or absence of 10 µM suramin. ATDC5s were differentiated 14 days (proliferation) in DMEM/F12 with insulin-transferrin-sodium selenite (ITS), and then 7 days (hypertrophy) in α-MEM with ITS, β-glycerophosphate and ascorbic acid. Readouts included analyses of extracellular matrix components (dimethylmethylene blue assay for sulfated glycosaminoglycan (sGAG) content, and histology), MMPs- and ADAMTS-induced aggrecan neo-epitopes (VDIPEN and NITEGE, by immunostaining) and phenotypic markers (quantitative PCR). We assessed the effects of suramin in vivo using the papain-induced model of osteoarthritis in C57BL/6 with or without a single intra-articular injection of 1 mg suramin. Cartilage damage was analysed by histology (OARSI scoring), and by Hexabrix contrast-enhanced nanofocus computed tomography, thereby quantifying cartilage volume.ResultsIn ATDC5 and hAC, TIMP-3 protein levels were increased. Presence of VDIPEN and NITEGE were decreased, thus sGAG content was increased. Noteworthy gene expression levels of phenotypic markers remained unaffected, suggesting a post-translational effect of suramin. Knees from Papain-triggered osteoarthritis in animals treated with suramin showed increased TIMP-3 and decreased VDIPEN and NITEGE levels. This potential protective mechanism effectively resulted in a decreased damage outcome and an increased cartilage volume.ConclusionOur data suggest that suramin has a protective effect by increasing the amount of TIMP3 in the articular cartilage, leading to higher sGAG levels. Therefore, suramin is a new potential osteoarthritic disease-modifying agent that appears to act specifically within the cartilage matrix.

Abnormal Wnt signaling is associated with bone mass disorders. Frizzled-related protein (FRZB, also known as secreted frizzled-related protein-3 (SFRP3)) is a Wnt modulator that contains an amino-terminal cysteine-rich domain (CRD) and a carboxy-terminal Netrin-like (NTN) motif. Frzb−/− mice show increased cortical thickness. However, the direct effect of FRZB on osteogenic differentiation and the involvement of the structural domains herein are not fully understood. In this study, we observed that stable overexpression of Frzb in MC3T3-E1 cells increased calcium deposition and osteoblast markers compared with control. Western blot analysis showed that the increased osteogenesis was associated with reduced canonical, but increased non-canonical Wnt signaling. On the contrary, loss of Frzb induced the opposite effects on osteogenesis and Wnt signaling. To translationally validate the positive effects of FRZB on primary human cells, we treated human periosteal and human bone marrow stromal cells with conditioned medium from MC3T3-E1 cells overexpressing Frzb and observed an increase in Alizarin red staining. We further studied the effect of the domains. FrzbNTN overexpression induced similar effects on osteogenesis as full-length Frzb, whereas FrzbCRD overexpressing cells mimicked loss of Frzb experiments. The CRD is considered as the Wnt binding domain, but the NTN domain also has important effects on bone biology. FRZB and other SFRPs or their specific domains may hold surprising potential as therapeutics for bone and joint disorders considering that excess of SFRPs has effects that are not expected under physiological, endogenous expression conditions.