DOT1L safeguards cartilage homeostasis and protects against osteoarthritis.
Ontology highlight
ABSTRACT:
SUBMITTER: Monteagudo S
PROVIDER: S-EPMC5481839 | biostudies-literature | 2017 Jun
REPOSITORIES: biostudies-literature
Publications
DOT1L safeguards cartilage homeostasis and protects against osteoarthritis.
Monteagudo Silvia S Cornelis Frederique M F FMF Aznar-Lopez Carolina C Yibmantasiri Ploi P Guns Laura-An LA Carmeliet Peter P Cailotto Frédéric F Lories Rik J RJ
Nature communications 20170619
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-activa ...[more]
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Project description: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.
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Project description:3'-Sialyllactose has specific physiological functions in a variety of tissues; however, its effects on osteoarthritic development remain unknown. Here, we demonstrated the function of 3'-sialyllactose on osteoarthritic cartilage destruction. In vitro and ex vivo, biochemical and histological analysis demonstrated that 3'-sialyllactose was sufficient to restore the synthesis of Col2a1 and accumulation of sulphated proteoglycan, a critical factor for cartilage regeneration in osteoarthritic development, and blocked the expression of Mmp3, Mmp13 and Cox2 induced by IL-1?, IL-6, IL-17 and TNF-?, which mediates cartilage degradation. Further, reporter gene assays revealed that the activity of Sox9 as a transcription factor for Col2a1 expression was accelerated by 3'-sialyllactose, whereas the direct binding of NF-?B to the Mmp3, Mmp13 and Cox2 promoters was reduced by 3'-sialyllactose in IL-1?-treated chondrocytes. Additionally, IL-1? induction of Erk phosphorylation and I?B degradation, representing a critical signal pathway for osteoarthritic development, was totally blocked by 3'-sialyllactose in a dose-dependent manner. In vivo, 3'-sialyllactose protected against osteoarthritic cartilage destruction in an osteoarthritis mouse model induced by destabilization of the medial meniscus, as demonstrated by histopathological analysis. Our results strongly suggest that 3'-sialyllactose may ameliorate osteoarthritic cartilage destruction by cartilage regeneration via promoting Col2a1 production and may inhibit cartilage degradation and inflammation by suppressing Mmp3, Mmp13 and Cox2 expression. The effects of 3'-sialyllactose could be attributed in part to its regulation of Sox9 or NF-?B and inhibition of Erk phosphorylation and I?B degradation. Taken together, these effects indicate that 3'-sialyllactose merits consideration as a natural therapeutic agent for protecting against osteoarthritis.
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Project description:Transforming growth factor-β (TGF-β) signaling is a critical regulator for articular cartilage tissue maintenance and chondrocyte homeostasis. Nonetheless, the regulatory networks and downstream signaling pathways that govern the chondroprotective function of TGF-β in the context of osteoarthritis (OA) are not fully defined. Recent studies reveal that mice with postnatal deletion of triple forkhead box class Os (FoxOs) (1, 3, and 4) spontaneously develop OA-like pathologies. The OA phenotype largely recapitulates that observed in mice with loss of TGF-βR2. In the present study, we investigated the role of FoxOs as downstream mediators of TGF-β signaling and define their role in articular cartilage homeostasis. Among the three FoxOs (1, 3, and 4), TGF-β signaling exclusively regulates FoxO1 in a TGF-β activated kinase 1 (TAK1)-dependent manner. Furthermore, FoxO1 was genetically ablated in mice in a tissue-specific manner in articular cartilage or overexpressed in adult cartilage immediately followed by meniscal/ligament injury (MLI). Histological and microcomputed tomography (micro-CT) analyses demonstrated that loss of FoxO1 postnatally in articular cartilage leads to OA-like pathologies, and gain of FoxO1 in adult cartilage has both preventative and therapeutic effects on surgically induced OA. Mechanistically, FoxO1 was found to maintain articular chondrocyte homeostasis through induction of anabolic and autophagy-related gene expressions. Importantly, overexpression of FoxO1 markedly rescued the OA phenotypes caused by deficiency in TGF-β signaling in chondrocytes. Our study identifies that TGF-β/TAK1-FoxO1 is a key signaling cascade in regulation of articular cartilage autophagy and homeostasis and is a potentially important therapeutic target for OA-like joint diseases.
| S-EPMC7720227 | biostudies-literature
Project description:ObjectiveOsteoarthritis (OA) is a progressive degenerative disease of the articular cartilage caused by an unbalanced activity of proteases, cytokines and other secreted proteins. Since heparan sulfate (HS) determines the activity of many extracellular factors, we investigated its role in OA progression.MethodsTo analyze the role of the HS level, OA was induced by anterior cruciate ligament transection (ACLT) in transgenic mice carrying a loss-of-function allele of Ext1 in clones of chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl). To study the impact of the HS sulfation pattern, OA was surgically induced in mice with a heterozygous (Ndst1+/-) or chondrocyte-specific (Col2-Cre;Ndst1fl/fl) loss-of-function allele of the sulfotransferase Ndst1. OA progression was evaluated using the OARSI scoring system. To investigate expression and activity of cartilage degrading proteases, femoral head explants of Ndst1+/- mutants were analyzed by qRT-PCR, Western Blot and gelatin zymography.ResultsAll investigated mouse strains showed reduced OA scores (Col2-rtTA-Cre;Ext1e2fl/e2fl: 0.83; 95% HDI 0.72-0.96; Ndst1+/-: 0.83, 95% HDI 0.74-0.9; Col2-Cre;Ndst1fl/fl: 0.87, 95% HDI 0.76-1). Using cartilage explant cultures of Ndst1 animals, we detected higher amounts of aggrecan degradation products in wildtype samples (NITEGE 4.24-fold, 95% HDI 1.05-18.55; VDIPEN 1.54-fold, 95% HDI 1.54-2.34). Accordingly, gelatin zymography revealed lower Mmp2 activity in mutant samples upon RA-treatment (0.77-fold, 95% HDI: 0.60-0.96). As expression of major proteases and their inhibitors was not altered, HS seems to regulate cartilage degeneration by affecting protease activity.ConclusionA decreased HS content or a reduced sulfation level protect against OA progression by regulating protease activity rather than expression.
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Project description:In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.
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Project description:Osteoarthritis (OA), characterized by chronic systemic low-level inflammation and cartilage degeneration, is a type of arthritis closely associated with aging. Inflammation and aging play a pivotal role in the occurrence and progression of OA. NLRP3 inflammasome is involved in many inflammatory and aging diseases, and NLRP3 inhibitor MCC950 has anti-inflammatory and antisenescence effects on some diseases such as Alzheimer's disease. In the present study, we found that NLRP3 protein was upregulated in human and mouse OA cartilage. Moreover, NLRP3 and Caspase1 expression induced by IL-1β in chondrocytes was blocked by MCC950. In addition, MCC950 inhibited the expression of inflammatory mediators, matrix-degrading enzymes, senescence marker protein P16 (INK4A), and β-galactosidase, as well as excessive production of ROS. Meanwhile, MCC950 promoted autophagy-related protein expression and autophagy flux under the inflammatory condition. However, autophagy inhibitor 3-MA reversed anti-inflammatory and anticatabolic effects of MCC950. In in vivo experiments, intra-articular administration of MCC950 further showed its protective effect on cartilage degeneration. Bioinformatic analysis and in vitro experimental results revealed that MCC950 might play a protective role in cartilage by regulating Nrf2/HO-1/NQO1, PI3k/Akt/mTOR, P38/MAPK, and JNK/MAPK pathways. In conclusion, our work demonstrated that NLRP3 inhibitor MCC950 might serve as a promising strategy for OA treatment.
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Project description:Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling and pain-causing properties. ER stress has been reported to be closely related to the progression of OA. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) pathway, which is highly expressed in the chondrocytes of OA patients, promotes the degradation and refolding of abnormal proteins during ER stress and maintains the stability of the ER environment of chondrocytes, but its function and the underlying mechanisms of how it contributes to the progression of OA remain unclear. This study investigates the role of IRE1α/ERN1 in OA. Specific deficiency of ERN1 in chondrocytes spontaneously resulted in OA-like cartilage destruction and accelerated OA progression in a surgically induced arthritis model. Local delivery of AdERN1 relieved degradation of the cartilage matrix and prevented OA development in an ACLT-mediated model. Mechanistically, progranulin (PGRN), an intracellular chaperone, binds to IRE1α, promoting its phosphorylation and splicing of XBP1u to generate XBP1s. XBP1s protects articular cartilage through TNF-α/ERK1/2 signaling and further maintains collagen homeostasis by regulating type II collagen expression. The chondroprotective effect of IRE1α/ERN1 is dependent on PGRN and XBP1s splicing. ERN1 deficiency accelerated cartilage degeneration in OA by reducing PGRN expression and XBP1s splicing, subsequently decreasing collagen II expression and triggering collagen structural abnormalities and an imbalance in collagen homeostasis. This study provides new insights into OA pathogenesis and the UPR and suggests that IRE1α/ERN1 may serve as a potential target for the treatment of joint degenerative diseases, including OA.
| S-EPMC10689778 | biostudies-literature
Project description:Osteoclasts are absorptive cells that play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role of epigenetic regulation in osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation (H3K79me), during osteoclast formation. Using RANKL-induced RAW264.7 macrophage cells as an osteoclast differentiation model, we found that DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. In addition, DOT1L inhibition increased osteoclast surface area and accelerated bone-mass reduction in a mouse ovariectomy (OVX) model of osteoporosis without alter osteoblast differentiation. DOT1L inhibition increase reactive oxygen species (ROS) generation and autophagy activity, and cell migration in pre-osteoclasts. Moreover, it strengthened expression of osteoclast fusion and resorption-related protein CD9 and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-regulated, H3K79me2-mediated, epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.
| S-EPMC5833786 | biostudies-literature
Project description:UnlabelledMicroRNAs 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.Key messagesExpression of the miR-29 family is regulated in cartilage during osteoarthritis. SOX9 represses expression of the miR-29 family in chondrocytes. The miR-29 family is regulated by TGF-β1 and IL-1 in chondrocytes. The miR-29 family negatively regulates Smad, NFκB, and canonical Wnt signalling. Several Wnt-related genes are direct targets of the miR-29 family.
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