Transcriptional deregulation during osteoclastic differentiation due to diuron exposure affects preferentially super-enhancers regulated genes
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ABSTRACT: Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. To better characterize the differentiation and maturation of CD14+ monocytes into functionally mature osteoclasts, we performed Chromatin Immunoprecipitation against H3K27ac followed by Sequencing (ChIP-Seq) and RNA-Sequencing at different stages of an in vitro differentiation model. The combinatorial study of the epigenetic and transcriptional dynamics taking place during the course of differentiation has allowed us to reveal a very dynamic epigenetic profile that supports the expression of genes vital for osteoclasts differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Since diuron, a commonly detected herbicide in our environment, is suspected to affect bone development, we then sought to evaluate the potential impact of diuron exposure on bone homeostasis. We performed RNA-Seq and functional test during in vitro osteoblastic and osteoclastic differentiation. First of all, our data suggest that high-dose of diuron (50 µM) affects viability of mesenchymal stem cells and thus inhibits bone mineralization. We observed at lower dose (1 µM) an inhibitory effect of this herbicide on number of osteoclasts derived from CD14+ monocytes without affecting cell viability. RNA-Seq data analysis revealed that diuron is specifically affecting the transcriptional network dedicated to osteoclasts differentiation and activity. Among the diuron affected genes, our analysis suggests a significant enrichment of genes targeted by gained super-enhancers along the differentiation process, with an odds ratio of 5.12 (ρ = 2.59 x 10-5). Taken together our results suggest that diuron exposure disrupts osteoclasts maturation by impairing the expression of cell-identity determining genes.
Project description:Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. Osteoclasts are responsible for bone resorption and play a vital role in this process. The role of salt-inducible kinase 3 (SIK3) in osteoclast differentiation and maturation and its potential as a therapeutic target for osteoporosis remain poorly understood. Here, we generated osteoclast-specific Sik3 conditional knockout (cKO) mice and investigated the effects of SIK3 deletion on bone homeostasis. Our findings revealed increased bone mineral density and an osteopetrosis phenotype in Sik3 cKO mice, indicating the involvement of SIK3 in bone resorption. Furthermore, we examined the effect of pterosin B, an SIK3 inhibitor, on osteoclast differentiation. Pterosin B treatment inhibits osteoclast differentiation in vitro, reduces the number of multinucleated osteoclasts, and suppresses bone resorption. Therefore, in this study, to better understand the molecular mechanism of SIK3 in osteoclast differentiation and maturation, we performed gene expression profiling of bone marrow monocytes from SIK3 deletion and pterosin B treated mice using RNA sequencing (RNA-seq).
Project description:Overactivated osteoclastogenesis leading to abnormal subchondral bone loss is the main feature of temporomandibular joint osteoarthritis (TMJOA). The role of N6-methyladenosine (m6A) in osteoclast differentiation in TMJOA remains unknown. Here, we found that an m6A reader IGF2BP2 was essential for mature osteoclasts induction. In TMJ tissues of TMJOA patients, the expression of IGF2BP2 was increased. Moreover, IGF2BP2 was augmented in subchondral bone of monosodium iodoacetate (MIA)–induced TMJOA mice. Igf2bp2 deficiency attenuated MIA-induced subchondral bone loss and suppressed osteoclast differentiation. Mechanistically, IGF2BP2 directly stabilized PPARγ and C-FOS mRNA to enhance the NFATC1 signaling, thereby inducing osteoclast maturation. Moreover, the stabilized PPARγ promoted the transcription of C-FOS, resulting in a further amplified signaling of NFATC1. In Igf2bp2-deficient cells, overexpression of PPARγ and C-FOS rescued the function of osteoclasts through restoring reduced levels of NFATC1. On the other hand, IGF2BP2/PPARγ/C-FOS axis facilitated the formation of osteoclasts by restoring the inhibited autophagy levels through the downregulation of ATG16L2. Using an IGF2BP2 inhibitor CWI1-2 hindered osteoclast formation and represented therapeutic effects for TMJOA. In summary, IGF2BP2 is essential in differentiation and maturation of osteoclasts and aggravates TMJOA via stabilizing PPARγ and C-FOS, which promoting NFATC1-mediated osteoclast signaling and ATG16L2-mediated autophagy.
Project description:Increasing evidences are revealing the important biological functions of apoptotic bodies (ABs). Here we identified the proteomic profiles of ABs from osteoclasts of distinct differentiation stages and investigated the potential functions. ABs were isolated from apoptotic bone marrow macrophages (BMMs), pre-osteoclasts (pOCs) or mature osteoclasts (mOCs). Proteomic signature analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by bioinformatic analysis showed that proteomic signatures of ABs exhibited high similarities with the parental cells. Functionally, pOC-ABs induced endothelial progenitor cell (EPC) differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via PDGF-BB. mOC-ABs induced mesenchymal stem cell (MSC) osteogenic differentiation and promoted bone formation via RANKL reverse signaling. Together, our results mapped the detailed proteomic landscapes of osteoclast derived ABs and demonstrated that the biological functions of ABs are largely inherited from corresponding parental cells, suggesting a novel extended intercellular regulation after cell apoptosis.
Project description:Carabrone covalently binds to Trx1at cysteine 73 and then inhibits enzymatic activity, which inhibits differentiation and induces apoptosis in osteoclasts. Carabrone prevents bone resorption in vivo and in vitro. Targeting Trx1 represents a promising target for the treatment of bone-resorptive diseases.
Project description:To understand the pathogenic mechanisms of bone erosion in rheumatoid arthritis (RA), we investigated osteoclast-specific epigenetic programs, RANKL-responsive super-enhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) in human osteoclasts.
Project description:To understand the pathogenic mechanisms of bone erosion in rheumatoid arthritis (RA), we investigated osteoclast-specific epigenetic programs, RANKL-responsive super-enhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) in human osteoclasts.
Project description:Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. To this end, human monocytes were isolated through a double-density gradient from healthy donor blood. The differentiation to macrophages was performed either in cell culture dishes or in Teflon-coated bags. The impact of these two distinct differentiation protocols on the gene expression profile of macrophages was assessed via RNA-Sequencing. The macrophages were then further differentiated to terminal osteoclasts through the addition of Receptor Activator of NF-κB Ligand (RANKL). The yield and function of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in Teflon-coated bags. Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.
Project description:Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel anti-osteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of the transcriptional reprogramming during human osteoclast differentiation. Osteoclasts were differentiated from CD14+-monocytes from eight female donors. RNA-sequencing during differentiation demonstrated 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors. These patterns showed distinct molecular functions, associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies, and bone mineral density SNPs. Network analyses showed mutual dependencies between the temporal expression patterns and provides insight into subtype-specific transcriptional networks. Donor specific expression patterns identified genes at monocyte stage, such as filamin B (FLNB) and oxidized low density lipoprotein receptor 1 (OLR1, encoding LOX-1), that are predictive for the resorptive activity of mature osteoclasts. Differentially expressed G-protein coupled receptors showed strong expression during osteoclast differentiation and associated with bone mineral density SNPs, implying a pivotal role in osteoclast differentiation and activity. The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5A receptor 1 (C5AR1), somatostatin receptor 2 (SSTR2), and free fatty acid receptor 4 (FFAR4/GPR120). Activating C5AR1 enhanced osteoclast formation, while activating SSTR2 decreased resorptive activity of mature osteoclasts, and activating FFAR4 decreased both number and resorptive activity of mature osteoclasts. In conclusion, we report the transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as anti-resorptive G-protein coupled receptors as well as FLNB and LOX-1 as potential molecular markers of osteoclast activity. These data can help future investigations to identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel anti-osteoporotic targets.
Project description:To understand the pathogenic mechanisms of bone erosion in rheumatoid arthritis (RA), we investigated osteoclast-specific epigenetic programs, RANKL-responsive super-enhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) in human osteoclasts. This dataset represent histone modification encoding active enhancers genome-wide.
Project description:To understand the pathogenic mechanisms of bone erosion in rheumatoid arthritis (RA), we investigated osteoclast-specific epigenetic programs, RANKL-responsive super-enhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) in human osteoclasts. In this dataset, we validated the presence of the predicted SE-eRNAs.