Abstract
Osteoclasts are the sole bone resorbing cells, which undertake opposing roles to osteoblasts to affect skeletal mass and structure. However, unraveling the comprehensive molecular mechanisms behind osteoclast differentiation is necessitated to overcome limitations and scarcity of available data, particularly in relation with the emerging roles of long non-coding RNAs (LncRNAs) in gene expression. In this study, we performed comprehensive and progressive analyses of the dynamic transcriptomes of murine osteoclasts, generated in vitro. We compared the total RNA-based transcriptomes of murine bone marrow derived cells with differentiated osteoclasts, while focusing on potentially novel genes and LncRNAs, to uncover critical genes and their associated pathways, which are differentially regulated during osteoclast differentiation. We found 4,214 differentially regulated genes during osteoclast differentiation, which included various types of LncRNAs. Among the upregulated protein coding genes not previously associated with osteoclast are Pheta1, Hagh, Gfpt1 and Nol4, while downregulated genes included Plau, Ltf, Sell and Zfp831. Notably, we report Nol4 as a novel gene related to osteoclast activity since Nol4 knockout mice Nol4 em1(International Mouse Phenotyping Consortium)J exhibit increased bone mineral density. Moreover, the differentially expressed LncRNAs included antisense and long intergenic non-coding RNAs, among others. Overall, immune-related and metabolism-related genes were downregulated, while anatomical morphogenesis and remodeling-related genes were upregulated in early-differentiated osteoclasts with sustained downregulation of immune-related genes in mature osteoclasts. The gene signatures and the comprehensive transcriptome of osteoclast differentiation provided herein can serve as an invaluable resource for deciphering gene dysregulation in osteoclast-related pathologic conditions.
Highlights
The balance between osteoclast and osteoblast activity can dictate pathogenesis of bone diseases
Bone marrow (BM) cells from C57 black 6 (CBL/6) mice were cultured in complete growth media (α-MEM supplemented with 10% fetal calf serum, penicillin/streptomycin and glutamine) and in the presence of soluble macrophage colonystimulating factor (M-CSF) (Prospec Technology, United Kingdom) at 100 ng/ml to generate bone marrow derived macrophages (BMDM)
The functional activity of generated osteoclasts was assessed by Osteo Assay, which showed statistically significant, distinct and large resorption areas with osteoclasts compared to osteoclast precursors and undifferentiated BMDM cells (Figures 1D,E)
Summary
The balance between osteoclast and osteoblast activity can dictate pathogenesis of bone diseases. Signaling via tumor necrosis factor (TNF)-family cytokine, receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and macrophage colonystimulating factor (M-CSF) are identified as the primary pathways associated with osteoclast differentiation (Asagiri and Takayanagi, 2007). Various genes including TNF receptor-associated factor (TRAF) 6, NFKB1, FOS, nuclear factor of activated T cells 1 (NFATC1), and dendritic cell-specific transmembrane protein (DC-STAMP) have been associated with osteoclastogenesis (Asagiri and Takayanagi, 2007), while the tartrate-resistant acid phosphatase (TRAcP) and cathepsin K (CTSK), released by osteoclasts during resorption are identified as specific osteoclast markers (Boyle et al, 2003; Kirstein et al, 2006). Amplified stimulation of osteoclastogenesis primarily via NFKB signaling can lead to increased osteolysis in osteoporosis (Novack and Teitelbaum, 2008)
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