Development of a simple osteoarthritis model useful to predict in vitro the anti-hypertrophic action of drugs

International audienceOsteoarthritis (OA) is characterized by cartilage degradation, inflammation, and hypertrophy. Therapies are mainly symptomatic and aim to manage pain. Consequently, medical community is waiting for new treatments able to reduce OA process. This study aims to develop an in vitro simple OA model useful to predict drug ability to reduce cartilage hypertrophy. Human primary OA chondrocytes were incubated with transforming growth factor beta 1 (TGF-β1). Hypertrophy was evaluated by Runx2, type X collagen, MMP13, and VEGF expression. Cartilage anabolism was investigated by Sox9, aggrecan, type II collagen, and glycosaminoglycan expression. In chondrocytes, TGF-β1 increased expression of hypertrophic genes and activated canonical WNT pathway, while it decreased dramatically cartilage anabolism, suggesting that this treatment could mimic some OA features in vitro. Additionally, EZH2 inhibition, that has been previously reported to decrease cartilage hypertrophy and reduce OA development in vivo, attenuated COL10A1 and MMP13 upregulation and SOX9 downregulation induced by TGF-β1 treatment. Similarly, pterosin B (an inhibitor of Sik3), and DMOG (a hypoxia-inducible factor prolyl hydroxylase which mimicks hypoxia), repressed the expression of hypertrophy markers in TGF-β stimulated chondrocytes. In conclusion, we established an innovative OA model in vitro. This cheap and simple model will be useful to quickly screen new drugs with potential anti-arthritic effects, in complementary to current inflammatory models, and should permit to accelerate development of efficient treatments against OA able to reduce cartilage hypertrophy

Antiproliferative effect of the histone demethylase inhibitor GSK-J4 in chondrosarcomas

International audienceChondrosarcoma (CS) is the second most common malignant bone sarcoma. Its treatment remains an issue, because this tumor is radio- and chemo-resistant. In the present study, we investigated the antitumoral potential of GSK-J4, a small molecule described as an inhibitor of histone demethylases UTX and JMJD3 (KDM6A and KDM6B), alone or in combination with cisplatin in CSs. Human CS-derived cell lines were treated with GSK-J4 in the presence or not of cisplatin. Survival curves were established and cell proliferation and cycle were evaluated by flow cytometry using dividing cell tracking technique utilizing carboxyfluorescein succinimidyl ester labeling, or DNA staining by propidium iodide. Apoptosis and senescence were also investigated. GSK-J4 decreased proliferation of CS cells. Additionally, it induced apoptosis in CH2879 and JJ012 cells, but not in SW1353 CSs. In addition, its association with cisplatin decreased cell proliferation more than drugs alone, whereas it did not increase apoptosis compared to cisplatin alone. Interestingly, GSK-J4 alone as well as in association with cisplatin did not affect chondrocyte survival or proliferation. In conclusion, this study suggests that demethylase inhibitors may be useful in improving therapy for CS in reducing its proliferation

Méthylation de H3K27 : régulation et rôle dans le cartilage articulaire

Osteoarthritis is the most widespread rheumatological disease and one of the main causes of pain and disability. This work aimed to study the role and regulation of the lysine 27 histone H3 tri-methylation (H3K27me3) during osteoarthritis and chondrogenesis.In the first part of the study, we showed that EZH2 inhibition by EPZ-6438 attenuates inflammation and metalloproteases release induced by IL-1β. EZH2 inhibition also reduces TGF-β1-mediated chondrocytes hypertrophy. EPZ-6438 attenuates in vivo cartilage degradation in osteoarthritis mice model. Furthermore, EZH2 inhibition ameliorates locomotor disability in mice and reduces NGF expression in chondrocytes.In the second part of the study, we highlighted that JMJD3 and UTX enhance mesenchymal stem cell chondrogenic differentiation. In addition, JMJD3 and UTX overexpression promotes cartilage formation and type II collagen production of mesenchymal stem cells after in vivo implantation.Our study demonstrated the importance of H3K27me3 in cartilage. While EZH2 is involved in inflammation, chondrocyte hypertrophy and cartilage degradation, JMJD3 and UTX enhance chondrogenesis, cartilage formation, and collagen production.L’arthrose est la maladie rhumatologique la plus répandue et une des causes majeures de douleur et de handicap. Au cours de ce travail nous avons étudié le rôle et la régulation de la tri-méthylation de la lysine 27 de l’histone H3 (H3K27me3) dans l’arthrose et la chondrogenèse.Dans une première partie, nous avons montré que l’inhibition de la méthylase EZH2 par l’EPZ-6438 atténue l’inflammation et la libération de métalloprotéases par les chondrocytes traités à l’IL-1β. L’inhibition d’EZH2 réduit également l’hypertrophie des chondrocytes induite par le TGF-β1. L’EZP-6438 attenue aussi la dégradation du cartilage in vivo dans un modèle d’arthrose murin. De plus, l’inhibition d’EZH2 diminue le handicap locomoteur chez la souris et réduit l’expression du NGF dans les chondrocytes.Dans une deuxième partie, nous avons mis en évidence que les déméthylases JMJD3 et UTX favorisent la différenciation chondrogénique des cellules souches mésenchymateuses. D’autre part, la surexpression de JMJD3 et UTX favorise la formation de cartilage et la production de collagène de type II des cellules souches mésenchymateuses après différenciation et implantation in vivo.Notre étude a montré l’importance de la marque H3K27me3 dans le cartilage. Alors qu’EZH2 est impliquée dans l’inflammation, l’hypertrophie des chondrocytes et la destruction du cartilage, JMJD3 et UTX favorisent la chondrogenèse, la formation du cartilage et la production de collagène

Méthylation de H3K27 : régulation et rôle dans le cartilage articulaire

Osteoarthritis is the most widespread rheumatological disease and one of the main causes of pain and disability. This work aimed to study the role and regulation of the lysine 27 histone H3 tri-methylation (H3K27me3) during osteoarthritis and chondrogenesis.In the first part of the study, we showed that EZH2 inhibition by EPZ-6438 attenuates inflammation and metalloproteases release induced by IL-1β. EZH2 inhibition also reduces TGF-β1-mediated chondrocytes hypertrophy. EPZ-6438 attenuates in vivo cartilage degradation in osteoarthritis mice model. Furthermore, EZH2 inhibition ameliorates locomotor disability in mice and reduces NGF expression in chondrocytes.In the second part of the study, we highlighted that JMJD3 and UTX enhance mesenchymal stem cell chondrogenic differentiation. In addition, JMJD3 and UTX overexpression promotes cartilage formation and type II collagen production of mesenchymal stem cells after in vivo implantation.Our study demonstrated the importance of H3K27me3 in cartilage. While EZH2 is involved in inflammation, chondrocyte hypertrophy and cartilage degradation, JMJD3 and UTX enhance chondrogenesis, cartilage formation, and collagen production.L’arthrose est la maladie rhumatologique la plus répandue et une des causes majeures de douleur et de handicap. Au cours de ce travail nous avons étudié le rôle et la régulation de la tri-méthylation de la lysine 27 de l’histone H3 (H3K27me3) dans l’arthrose et la chondrogenèse.Dans une première partie, nous avons montré que l’inhibition de la méthylase EZH2 par l’EPZ-6438 atténue l’inflammation et la libération de métalloprotéases par les chondrocytes traités à l’IL-1β. L’inhibition d’EZH2 réduit également l’hypertrophie des chondrocytes induite par le TGF-β1. L’EZP-6438 attenue aussi la dégradation du cartilage in vivo dans un modèle d’arthrose murin. De plus, l’inhibition d’EZH2 diminue le handicap locomoteur chez la souris et réduit l’expression du NGF dans les chondrocytes.Dans une deuxième partie, nous avons mis en évidence que les déméthylases JMJD3 et UTX favorisent la différenciation chondrogénique des cellules souches mésenchymateuses. D’autre part, la surexpression de JMJD3 et UTX favorise la formation de cartilage et la production de collagène de type II des cellules souches mésenchymateuses après différenciation et implantation in vivo.Notre étude a montré l’importance de la marque H3K27me3 dans le cartilage. Alors qu’EZH2 est impliquée dans l’inflammation, l’hypertrophie des chondrocytes et la destruction du cartilage, JMJD3 et UTX favorisent la chondrogenèse, la formation du cartilage et la production de collagène

H3K27 methylation : regulation and role in articular cartilage

L’arthrose est la maladie rhumatologique la plus répandue et une des causes majeures de douleur et de handicap. Au cours de ce travail nous avons étudié le rôle et la régulation de la tri-méthylation de la lysine 27 de l’histone H3 (H3K27me3) dans l’arthrose et la chondrogenèse.Dans une première partie, nous avons montré que l’inhibition de la méthylase EZH2 par l’EPZ-6438 atténue l’inflammation et la libération de métalloprotéases par les chondrocytes traités à l’IL-1β. L’inhibition d’EZH2 réduit également l’hypertrophie des chondrocytes induite par le TGF-β1. L’EZP-6438 attenue aussi la dégradation du cartilage in vivo dans un modèle d’arthrose murin. De plus, l’inhibition d’EZH2 diminue le handicap locomoteur chez la souris et réduit l’expression du NGF dans les chondrocytes.Dans une deuxième partie, nous avons mis en évidence que les déméthylases JMJD3 et UTX favorisent la différenciation chondrogénique des cellules souches mésenchymateuses. D’autre part, la surexpression de JMJD3 et UTX favorise la formation de cartilage et la production de collagène de type II des cellules souches mésenchymateuses après différenciation et implantation in vivo.Notre étude a montré l’importance de la marque H3K27me3 dans le cartilage. Alors qu’EZH2 est impliquée dans l’inflammation, l’hypertrophie des chondrocytes et la destruction du cartilage, JMJD3 et UTX favorisent la chondrogenèse, la formation du cartilage et la production de collagène.Osteoarthritis is the most widespread rheumatological disease and one of the main causes of pain and disability. This work aimed to study the role and regulation of the lysine 27 histone H3 tri-methylation (H3K27me3) during osteoarthritis and chondrogenesis.In the first part of the study, we showed that EZH2 inhibition by EPZ-6438 attenuates inflammation and metalloproteases release induced by IL-1β. EZH2 inhibition also reduces TGF-β1-mediated chondrocytes hypertrophy. EPZ-6438 attenuates in vivo cartilage degradation in osteoarthritis mice model. Furthermore, EZH2 inhibition ameliorates locomotor disability in mice and reduces NGF expression in chondrocytes.In the second part of the study, we highlighted that JMJD3 and UTX enhance mesenchymal stem cell chondrogenic differentiation. In addition, JMJD3 and UTX overexpression promotes cartilage formation and type II collagen production of mesenchymal stem cells after in vivo implantation.Our study demonstrated the importance of H3K27me3 in cartilage. While EZH2 is involved in inflammation, chondrocyte hypertrophy and cartilage degradation, JMJD3 and UTX enhance chondrogenesis, cartilage formation, and collagen production

Epigenetic dynamic during endochondral ossification and articular cartilage development

International audienceWithin the last decade epigenetics has emerged as fundamental regulator of numerous cellular processes, including those orchestrating embryonic and fetal development. As such, epigenetic factors play especially crucial roles in endochondral ossification, the process by which bone tissue is created, as well during articular cartilage formation. In this review, we summarize the recent discoveries that characterize how DNA methylation, histone post-translational modifications and non-coding RNA (e.g., miRNA and lcnRNA) epigenetically regulate endochondral ossification and chondrogenesis

Anti-inflammatory and chondroprotective effects of the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A, in human articular chondrocytes

International audience3-Deazaneplanocin A (DZNep) is an inhibitor of S-Adenosyl-L-Homocysteine Hydrolase (SAHH) known to inhibit EZH2, a histone methylase upregulated during osteoarthritis. In this study, we assessed its effects in human articular chondrocytes. Anti-inflammatory effects were assessed by Nitric Oxide (NO), Prostaglandin E2 (PGE2) and Metalloprotease (MMP) release in IL-1β-stimulated chondrocytes. MAPK and NFκB activation was analyzed by western blotting. Differentially expressed genes (DEG) regulated by DZNep were identified by whole-transcriptome microarray. DZNep inhibited SAHH activity and was not toxic. It counteracted NO, PGE2 and MMP release, and reduced MAPK activation induced by IL-1β. By whole-transcriptome analysis, we identified that DNZep counteracts the effect of IL-1β on the expression of 81 protein-coding genes, including CITED2, an MMP inhibitor. These genes are organized in a protein-protein network centred on EGR1, which is known to functionally interact with EZH2. Gene ontologies enrichment analysis confirmed that DZNep counteracts IL-1β-induced expression of genes involved in cartilage matrix breakdown (MMPs and ADAMTS). In addition, DZNep up-regulated cartilage specific genes, such as COL2A1 and SOX9, suggesting a chondroprotective effect of DZNep. DZNep exhibits anti-inflammatory effects, and regulates genes implicated in chondroprotective response in human articular chondrocytes, suggesting that inhibitors of S-adenosylmethionine-dependent methyltransferases could be effective treatments for OA

EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis

International audienceHistone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1β. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1β in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1β effects. Ex vivo, EZH2 inhibition decreased IL-1β-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain. Abbreviations DNZep 3-Deazanoplanocin A EZH2 Enhancer of Zest Homolog 2 GFP Green Fluorescent Protein H3K27 Lysine 27 of the histone 3 IL-1β Interleukin-1β IL-6 Interleukin 6 JMJD3 Jumonji Domain Containing 3 MMP Matrix metalloproteinase MMX Medial meniscectomy NGF Nerve Growth Factor NO Nitric oxide OA Osteoarthritis PGE2 Prostaglandin E2 Osteoarthritis (OA) is the most widespread rheumatic disease worldwide, and one of the main causes of pain and disability, reducing patient's quality of life 1. OA affects 1 in 3 people over age 65 and women more than men 2. In the United States, 22.7% of the adult population (52.5 million of persons) report having been diagnosed with OA by their physician. The impact on healthcare expenses is massive. The cost of knee OA alone is, for instance