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A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
Comprehensive Transcriptomic Profiling of Murine Osteoclast Differentiation Reveals Novel Differentially Expressed
Salman M Toor1, Sachin Wani2, Omar M E Albagha1,2
1College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar.
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 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.
Insights
This study reveals novel genes and long non-coding RNAs (LncRNAs) involved in osteoclast differentiation. Identifying these molecular regulators, like Nol4, aids understanding bone remodeling and related diseases.
Area of Science:
- Molecular Biology
- Genetics
- Bone Biology
Background:
- Osteoclasts are crucial for bone resorption, balancing osteoblast activity to maintain skeletal integrity.
- Understanding osteoclast differentiation mechanisms is vital for addressing bone diseases.
- Long non-coding RNAs (LncRNAs) are increasingly recognized for their roles in gene regulation.
Purpose of the Study:
- To comprehensively analyze the dynamic transcriptomes of murine osteoclasts during in vitro differentiation.
- To identify novel protein-coding genes and LncRNAs differentially regulated during osteoclastogenesis.
- To uncover critical genes and pathways involved in osteoclast differentiation and bone remodeling.
Main Methods:
- Comparative transcriptome analysis of murine bone marrow-derived cells and differentiated osteoclasts.
- Focus on identifying novel genes and LncRNAs using total RNA sequencing.
- Bioinformatic analysis to determine differentially expressed genes and associated pathways.
Main Results:
- Identified 4,214 differentially regulated genes during osteoclast differentiation, including various LncRNAs.
- Discovered novel upregulated genes (e.g., Pheta1, Hagh, Gfpt1, Nol4) and downregulated genes (e.g., Plau, Ltf, Sell, Zfp831).
- Reported Nol4 as a novel gene associated with osteoclast activity, with knockout mice showing increased bone mineral density.
Conclusions:
- Osteoclast differentiation involves significant changes in gene expression, including novel LncRNAs.
- Specific genes like Nol4 play critical roles in bone homeostasis.
- The study provides a valuable transcriptomic resource for investigating osteoclast-related pathologies.
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