Silvia Monteagudo

DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

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.

Cushioning the cartilage: a canonical Wnt restricting matter.

Wnt signalling pathways have key roles in joint development, homeostasis and disease, particularly in osteoarthritis. New data is starting to reveal the importance of tightly regulating canonical Wnt signalling pathway activation to maintain homeostasis and health in articular cartilage. In addition to the presence of different Wnt antagonists that limit pathway activation in articular cartilage, the reciprocal crosstalk between the canonical and non-canonical cascades and competitive antagonism between different Wnt ligands seem to be critical in restraining excessive Wnt pathway activation. Changes in transcriptional complex assembly upon Wnt pathway activation, epigenetic modulation of target gene transcription, in particular through histone modifications, and complex interactions between the Wnt signalling pathway and other signalling pathways, are also instrumental in adjusting Wnt signalling. In this Review, the cellular and molecular mechanisms involved in fine-tuning canonical Wnt signalling in the joint are updated, with a focus on the articular cartilage. The interventions for preventing or treating osteoarthritis are also discussed, which should aim to limit disease-associated excessive canonical Wnt activity to avoid joint damage.

ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone

ANP32A is a transcriptional regulator of ATM and provides protection against oxidative stress to prevent joint, brain, and bone disease. Oxidative stress and osteoarthritis Osteoarthritis is a common degenerative joint disorder that affects cartilage and bone. Cornelis et al. investigated the role of ANP32A, a protein involved in multiple cellular processes, in osteoarthritis. ANP32A was decreased in osteoarthritic human and mouse tissue samples and also decreased with aging. The authors found that ANP32A promoted transcription of ATM and regulated reactive oxygen species in cartilage. Antioxidant therapy protected Anp32a-deficient mice from developing osteoarthritis and osteopenia and also rescued neurological defects caused by lack of ATM and increased oxidative stress. These results suggest that ANP32A could be a therapeutic target for correcting imbalanced reactive oxygen species and antioxidants. 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 Anp32a-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.

SMOC2 inhibits calcification of osteoprogenitor and endothelial cells

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.

A Notch in the joint that exacerbates osteoarthritis

Tight regulation of signalling cascades is vital for the correct development and function of bones and joints. A new study suggests that Notch signalling might join the likes of the transforming growth factor superfamily and Wnt signalling cascades as having an important function in joint homeostasis and disease.

Wnt signaling as target for the treatment of osteoarthritis

Osteoarthritis is a severe and common rheumatic and skeletal disease for which currently no specific drugs are available. The Wnt signaling pathway modulates key biological processes in development, growth, homeostasis, and disease, particularly in the joints and bone. Excessive activation of the Wnt signaling pathway in the articular cartilage has been clearly associated with the onset and severity of osteoarthritis. Hence, targeting Wnt signaling may be an excellent approach to develop specific drugs useful for the treatment of osteoarthritis. In this article, we review the biology of Wnt signaling in the context of osteoarthritis; we also analyze the gradual improvement of our molecular understanding of Wnts in the joint and oversee current progress toward the development of Wnt inhibition as therapy for osteoarthritis. At least one Wnt inhibitor is currently going forward in the clinical evaluation process, potentially marking the beginning of a new era in the management of osteoarthritis.

Suramin increases cartilage proteoglycan accumulation in vitro and protects against joint damage triggered by papain injection in mouse knees in vivo

Objectives Suramin 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). Methods In 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. Results In 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. Conclusion Suramin 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.

SP0019 Histone Modification and Osteoarthritis

Epigenetic mechanisms, processes that alter gene expression without changes in DNA sequence, precisely regulate gene expression by dynamic remodeling of chromatin. Eukaryotic DNA is wrapped around a histone octamer (H3/H4 heterotetramer and two H2A/H2B dimers) to form the nucleosome, the fundamental building block of chromatin. Histone proteins are subject to covalent modifications that modulate the local structure of chromatin and influence gene expression by regulating the accessibility of gene loci to transcriptional machinery. The dynamic remodeling of chromatin through histone modifications by nuclear proteins including histone acetyltransferases, deacetylases, and methyltransferases, as well as by demethylases, modulate spatiotemporal gene expression during cartilage development, homeostasis and disease. Disruptor of telomeric silencing 1-like (DOT1L) is the main histone-modifying enzyme that catalyzes the methylation on lysine-79 of histone 3 (H3K79). The human protein DOT1L contains 1537 aminoacids, with its N-terminal part responsible for the methyltransferase activity. The remaining C-terminal part is involved in physical interactions with many transcription relevant proteins. The general function of DOT1L is to methylate H3K79 as a member of a large protein complex, which can influence the transcriptional state. DOT1Ls histone methyltransferase activity has been linked to active transcription, and plays a role in many biological processes including the DNA damage response, the cell cycle, embryonic development and cell reprogramming. In cancer biology, DOT1L is involved in MLL-rearranged leukemia and in other tumors. We reported a strong genetic association between polymorphisms in the human DOT1L gene and hip cartilage thickness as well as osteoarthritis (OA). Variations in the DOT1L gene are also associated with human height. Furthermore, we earlier demonstrated that silencing of Dot1L inhibited chondrogenic differentiation of murine progenitor cells. Interestingly, DOT1L-associated H3K79 methylation has been linked to the Wingless-type (Wnt) cascade. Gain and loss of function studies for Wnt signaling mediator beta-catenin or Wnt antagonists such as Frizzled related protein have demonstrated that tight regulation of this pathway is key to joint health. This could specifically identify DOT1L as an attractive target for joint diseases, more particularly for OA. DOT1L is an enzyme and, thus, can be pharmacologically modulated. We therefore hypothesized that DOT1L plays a major role in cartilage homeostasis by tightly regulating gene expression patterns of key signaling pathways, such as the Wnt cascade. New insights into the transcriptional mechanisms may contribute to the development of new epigenetics-based strategies for the treatment of joint diseases, adding a new way to modulate fundamental signaling pathways in joint disease and repair. Disclosure of Interest S. Monteagudo Grant/research support from: Marie Curie Fellowship

A8.04 The histone methyltransferase dot1l is essential for growth and homeostasis of the articular cartilage

Background and objectives DOT1L is the only known H3K79 histone methyltransferase. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signalling. Variations in the DOT1L gene are also associated with human height. These findings and our earlier in vitro insights prompted us to investigate the impact of DOT1L loss of function in vivo. Methods We generated a conditional cartilage-specific knockout (KO) model of DOT1L by crossing Dot1lfl/fl mice with Col2-Cre+/- mice. The deletion of exon2 in the Dot1l gene in cartilage was confirmed by PCR. We performed skeletal staining to identify skeletal abnormalities and growth defects, and histology of the growth plate. To study if the absence of DOT1L activity causes the development of early OA, we injected the chemical DOT1L inhibitor EPZ-5676 intra-articularly in the right knee of 8-week-old wild-type C57Bl/6 mice. Severity of disease was determined by histological scores on sections throughout the knee. Both cartilage damage and synovial hyperplasia were assessed based on OARSI guidelines. Based in our in vitro observations, we analysed the effect of DOT1L loss of function on specific Wnt target genes in either DOT1L cartilage-specific KO mice or in mice injected with EPZ-5676, by immunohistochemistry. Results A growth defect resulting in short stature became quickly apparent and resulted in mortality from the age of 4-weeks onwards in Dot1l cartilage-specific KO mice. These DOT1L deficient mice exhibited changes in the growth plates with a reduced and disorganised proliferative and pre-hypertrophic zone. In mice injected with EPZ-5676, histology severity scores were significantly increased over time compared to control treatment groups. We confirmed the regulatory role of DOT1L on canonical Wnt signalling in cartilage, since the absence of DOT1L activity in either cartilage specific KO mice or mice injected with DOT1L inhibitor resulted in increased protein levels of specific Wnt target genes. Conclusions Our findings support an essential role for DOT1L in growth and cartilage homeostasis as a key regulator of canonical WNT signalling in the joint.

A4.13 The DOT1L protein and gene network in chondrocytes identifies H3K79 histone methylation as key regulator of WNT and other growth factor cascades

Background and objectives DOT1L is the only known H3K79 histone methyltransferase. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signalling. Here, we further investigated the biology of DOT1L in cartilage health and disease. Specifically, our objective was to define the transcriptional complexes and transcriptome associated with DOT1L in articular cartilage, and to investigate the role of DOT1L during the loss of phenotype of the articular chondrocyte. Methods Human articular chondrocytes (hACs) stimulated or not with LiCl were used to carefully map the presence or absence of DOT1L protein complexes suggested earlier in leukaemia cells, using immunoprecipitation experiments. To elucidate the transcriptional network of DOT1L, we performed a microarray of hACs from 5 non-OA fracture patients treated with a specific DOT1L inhibitor (EPZ5676) or vehicle. To establish the role of DOT1L in the maintenance of hACs phenotype, hACs were cultured in monolayer in the presence or absence of EPZ5676, and RNA and protein were isolated at serial passages. We analysed by quantitative PCR the gene expression of known genes important for chondrocyte biology and/or genes that appeared in the microarray of DOT1L inhibition. Activation of the Wnt canonical and non-canonical signalling cascades was analysed by Western blot. Results The presence of different DOT1L elongation complexes was confirmed in hACs. In the microarray, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated in EPZ5676 treated samples; OA-related genes, such as MMP1, CCL7, CCL8 and GPNMB were up-regulated; and WNT target genes, ligands and antagonists such as LEF1, WNT5A and DKK1 were significantly up-regulated. In the de-differentiation experiment, genes involved in relevant pathways for cartilage biology such as the WNT, NOTCH and BMP pathways exhibited significant changes that were highly accentuated by DOT1L inhibition. Conclusions Our transcriptomic, protein and gene interaction approach provides novel insights into the DOT1L molecular network and its putative role in osteoarthritis and cartilage. These data further support an important role for DOT1L in joint homeostasis as a key regulator of WNT signalling and other growth factor cascades in the joint.