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Updated: Jun 8, 2025
Osteoclast Derivation from Mouse Bone Marrow
Published on: November 6, 2014
Extracellular vesicles from mouse bone marrow macrophages-derived osteoclasts treated with zoledronic acid contain
Sakura Minami1, Yasuyuki Fujii2, Yusuke Yoshioka3
1Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan; Department of Molecular and Cellular Medicine, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.
Abstract:
Medication-related osteonecrosis of the jaw (MRONJ) is an intractable form of osteonecrosis of the jaw that rarely occurs in patients using bone resorption inhibitors such as bisphosphonates (BPs). Then, extracellular vesicles (EVs) carry various signaling molecules, such as mRNAs, microRNAs (miRNAs), and proteins, and have attracted attention as intercellular communication tools. Recently, the role of EVs in communication between osteoclasts and surrounding bone cells has been confirmed. This study aimed to elucidate the effects of EVs derived from osteoclasts treated with zoledronic acid (ZA), one of the BPs on osteoclast function. EVs were isolated by ultracentrifugation of the culture supernatant of osteoclasts treated with ZA, and miRNAs were extracted from these EVs. Tartrate-resistant acid phosphatase staining of the ZA treated osteoclasts showed reduced osteoclastogenesis. In addition, pit assay showed that ZA significantly decreased the bone resorption capacity of osteoclasts. miRNA-seq analysis identified 11 upregulated and 5 downregulated differentially expressed genes (DEGs) in the miRNA of EVs derived from ZA-treated osteoclasts compared to EVs derived from osteoclasts not treated with ZA. qRT-PCR analysis confirmed the amount of these specific miRNAs, with miR-146a-5p, and miR-322-3p being significantly upregulated by ZA. Overexpression of miR-146a-5p in osteoclasts inhibited osteoclastogenesis and decreased the mRNA expression of osteoclast markers. In addition, Traf6 was identified as a candidate target gene of miR-146a-5p in several miRNA databases. Indeed, the overexpression of miR-146a-5p decreased the expression level of Traf6 in osteoclasts. Additionally, overexpression of miR-322-3p in the pre-osteoblast, MC3T3-E1 cells, resulted in a significant increase in the mRNA expression levels of Sp7. Our data indicate that BPs attenuate osteoclastogenesis by simultaneously altering the characteristics of osteoclast-derived EVs. Overexpression of miR-146a-5p and miR-322-3p influences osteoclast differentiation, and Traf6 is a target gene of miR-146a-5p. On the other hand, Overexpression of miR-322-3p affects osteoblast differentiation. We suggest that ZA-treated osteoclast-derived EVs may play an important role in osteoclast function and bone resorption.
Insights
Bisphosphonates (BPs) alter osteoclast-derived extracellular vesicles (EVs), impacting bone resorption. Specific microRNAs (miRNAs) within these EVs, like miR-146a-5p, influence osteoclast function and Traf6 expression, potentially mediating BP effects in medication-related osteonecrosis of the jaw (MRONJ).
Area of Science:
- Biochemistry
- Cell Biology
- Oral Biology
Background:
- Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication associated with bone resorption inhibitors like bisphosphonates (BPs).
- Extracellular vesicles (EVs) mediate intercellular communication, carrying signaling molecules such as microRNAs (miRNAs).
- The role of EVs in bone cell communication, particularly between osteoclasts, is increasingly recognized.
Purpose of the Study:
- To investigate the impact of zoledronic acid (ZA), a bisphosphonate, on osteoclast-derived EVs.
- To identify specific miRNAs within ZA-treated osteoclast EVs and their functional consequences.
- To elucidate the mechanisms by which BPs influence osteoclast function via EVs.
Main Methods:
- Osteoclasts were treated with zoledronic acid (ZA).
- Extracellular vesicles (EVs) were isolated from culture supernatants via ultracentrifugation.
- miRNA sequencing (miRNA-seq) and quantitative real-time PCR (qRT-PCR) were used to analyze miRNA profiles.
- Functional assays, including tartrate-resistant acid phosphatase staining, pit assays, and gene/protein expression analysis, were performed.
Main Results:
- ZA treatment reduced osteoclastogenesis and bone resorption capacity.
- miRNA-seq identified significant upregulation of miR-146a-5p and miR-322-3p in EVs from ZA-treated osteoclasts.
- Overexpression of miR-146a-5p inhibited osteoclastogenesis and decreased Traf6 mRNA expression.
- Overexpression of miR-322-3p increased Sp7 mRNA expression in pre-osteoblast cells.
Conclusions:
- Bisphosphonates attenuate osteoclastogenesis by altering the miRNA content of osteoclast-derived EVs.
- miR-146a-5p and miR-322-3p play key roles in regulating osteoclast and osteoblast differentiation, respectively.
- ZA-treated osteoclast-derived EVs, particularly via altered miRNA profiles, are implicated in modulating bone cell function and resorption.
