Osteoarthritis is the most common joint disorder with increasing global prevalence due to aging of the population. Current therapy is limited to symptom relief, yet there is no cure. Its multifactorial etiology includes oxidative stress and overproduction of reactive oxygen species, but the regulation of these processes in the joint is insufficiently understood. We report that ANP32A protects the cartilage against oxidative stress, preventing osteoarthritis development and disease progression. ANP32A is down-regulated in human and mouse osteoarthritic cartilage. Microarray profiling revealed that ANP32A protects the joint by promoting the expression of ATM, a key regulator of the cellular oxidative defense. Antioxidant treatment reduced the severity of osteoarthritis, osteopenia, and cerebellar ataxia features in -deficient mice, revealing that the ANP32A/ATM axis discovered in cartilage is also present in brain and bone. Our findings indicate that modulating ANP32A signaling could help manage oxidative stress in cartilage, brain, and bone with therapeutic implications for osteoarthritis, neurological disease, and osteoporosis.

Mice subjected to a running regime have significant increased cartilage damage and synovitis scores. Lack of Dio2 protected against cartilage damage in this model and was reflected in a specific gene expression profile, and either mark a favourable effect in the Dio2 knockout (eg, Gnas) or an unfavourable effect in wild-type cartilage homeostasis (eg, Hmbg2 and Calr). These data further support DIO2 activity as a therapeutic target in OA.

Objective: We earlier identified that the histone methyltransferase Disruptor of telomeric silencing 1-like (DOT1L) is as a master protector of cartilage health via limiting excessive activation of the Wnt pathway. However, cartilage-specific homozygous Dot1l knockout mice exhibited a severe growth phenotype and perinatal death, which hampered their use in induced or ageing models of osteoarthritis (OA). The aim of this study was to generate and examine haploinsufficient and inducible conditional Dot1l-deficient mouse models to evaluate the importance of DOT1L during post-traumatic or ageing-associated OA onset and progression. Method: We used cartilage-specific heterozygous and postnatal tamoxifen-inducible Dot1l knockout mice and performed destabilization of the medial meniscus (DMM) and ageing as OA models. Mice were examined histologically using X-rays and micro-computed tomography (mCT), and cartilage damage and osteophyte formation were assessed based on OARSI guidelines. Immunohistochemistry of DOT1L, H3K79me2, TCF1 and COLX was performed. Results: Both Dot1l-deficient strains exhibit a phenotype characterized by joint remodeling with extensive osteophyte formation and ectopic ossification upon ageing, indicating accelerated development of spontaneous osteoarthritis. In the DMM-induced OA mouse model, absence of Dot1l resulted in increased cartilage damage. Wnt signalling hyper-activation and ectopic chondrocyte hypertrophy were observed in the articular cartilage of both Dot1l-deficient mice. Conclusions: This study demonstrated the functional relevance of DOT1L in vivo during the development of OA using genetically modified mice. Thus, maintaining or enhancing DOT1L activity during ageing or after trauma might prevent OA onset and progression.

is further reinforced by the increased expression of apoptotic and autophagic markers in the articular cartilage of Klotho-deficient mice, and the increasing number of studies demonstrating that excessive induction of autophagy in OA-chondrocytes led to cell death by apoptosis. Further work is needed to decipher the link between autophagy and apoptosis in the pathogenesis of OA in order to develop new treatment to prevent chondrocyte death.

ObjectivesIn osteoarthritis, methylation of lysine 79 on histone H3 (H3K79me), a protective epigenetic mechanism, is reduced. Histone methylation levels are dynamically regulated by histone methyltransferases and demethylases. Here, we aimed to identify which histone demethylases regulate H3K79me in cartilage and investigate whether their targeting protects against osteoarthritis.MethodsWe determined histone demethylase expression in human non-osteoarthritis and osteoarthritis cartilage using qPCR. The role of histone demethylase families and subfamilies on H3K79me was interrogated by treatment of human C28/I2 chondrocytes with pharmacological inhibitors, followed by western blot and immunofluorescence. We performed C28/I2 micromasses to evaluate effects on glycosaminoglycans by Alcian blue staining. Changes in H3K79me after destabilisation of the medial meniscus (DMM) in mice were determined by immunohistochemistry. Daminozide, a KDM2/7 subfamily inhibitor, was intra-articularly injected in mice upon DMM. Histone demethylases targeted by daminozide were individually silenced in chondrocytes to dissect their role on H3K79me and osteoarthritis.ResultsWe documented the expression signature of histone demethylases in human non-osteoarthritis and osteoarthritis articular cartilage. Inhibition of Jumonji-C demethylase family increased H3K79me in human chondrocytes. Blockade of KDM2/7 histone demethylases with daminozide increased H3K79me and glycosaminoglycans. In mouse articular cartilage, H3K79me decayed rapidly upon induction of joint injury. Early and sustained intra-articular treatment with daminozide enhanced H3K79me and exerted protective effects in mice upon DMM. Individual silencing of KDM7A/B demethylases in human chondrocytes demonstrated that KDM7A/B mediate protective effects of daminozide on H3K79me and osteoarthritis.ConclusionTargeting KDM7A/B histone demethylases could be an attractive strategy to protect joints against osteoarthritis.

ObjectiveTo identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis.MethodsPreviously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition.ResultsDifferential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > |1,5|) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.ConclusionWe here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

No abstract

Purpose: Understanding the molecular mechanisms involved in cartilage health and disease is essential for the development of effective therapies for osteoarthritis (OA), of which there is a large unmet medical need. We earlier discovered that chemical inhibition of histone methyltransferase DOT1L triggers osteoarthritis in mouse knees, thus identifying DOT1L as a master protector of cartilage health. We also demonstrated that DOT1L protects the joint against the development of OA by limiting excessive activation of the Wnt pathway, a cascade that when hyper-activated can lead to joint disease. However, constitutive cartilage-specific homozygous Dot1l knockout mice used in this previous study exhibited a severe growth phenotype that hampered their use in induced or ageing models of OA, pointing out the need of using haplo-insufficient or inducible conditional mouse models. The aim of this novel study was to further understand the role of endogenous DOT1L in translational models of post-traumatic or ageing-associated OA. Methods: We used cartilage-specific Dot1l heterozygous knockout mice (Dot1l gene was targeted by Col2a1-Cre during developmental chondrogenesis) and conditional tamoxifen-induced cartilage-specific Dot1l knockout mice (Dot1l gene was targeted by Acan-Cre postnatally, at week 8). The effect on cartilage loss and eventual spontaneous OA was studied using X-rays, mCT, and histomorphological analysis of Safranin O stained sections from knees. In the postnatal Dot1l loss of function model, we also induced OA by destabilization of the medial meniscus (DMM). Both cartilage damage and synovial hyperplasia were assessed based on OARSI guidelines. Immunohistochemistry of COLX (marker of hypertrophic chondrocyte differentiation) and H3K79me2 (marker of DOT1L methyltransferase activity) were performed in all mouse models. Results: H3K79 methylation was partially reduced in haplo-insufficient Dot1l cartilage-specific knockout mice and strongly reduced in tamoxifen-induced Dot1l cartilage-specific knockout mice. Both Dot1l-deficient strains exhibit a phenotype characterized by joint remodeling with extensive osteophyte formation and ectopic ossification upon ageing, indicating accelerated development of spontaneous osteoarthritis. In the DMM-induced OA mouse model, induced cartilagespecific absence of Dot1l resulted in increased cartilage damage and enhanced ectopic chondrocyte hypertrophy in the articular cartilage, as compared to control animals.Conclusions: This study further validates the role of endogenous DOT1L and H3K79 methylation as a protector of cartilage health, and demonstrates that the DOT1L network may be a potential target for therapeutic intervention in ageing and post-traumatic OA.Purpose: Osteoarthritis (OA) is a disabling disease that affects more than 250 million people worldwide causing tremendous individual and socioeconomic burden. OA is a complex disease encompassing multiple molecular/biological mechanisms that result in progressive cartilage loss, subchondral bone remodeling, joint tis...