Dataset Information

Comparative characterization of osteoclasts derived from murine bone marrow macrophages and RAW 264.7 cells using quantitative proteomics

ABSTRACT: Osteoclasts (OCs) are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to M-CSF and RANKL. In vitro models are principally based on primary bone marrow macrophages, but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form OCs in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in OCs derived from bone marrow macrophages and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different; including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major differences between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line when studying M-CSF-dependent signaling and functions.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Osteoclast, Bone Marrow, Macrophage

SUBMITTER: Andrew Ng

LAB HEAD: Shuying Yang

PROVIDER: PXD009610 | Pride | 2018-11-27

REPOSITORIES: Pride

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Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics.

Ng Andrew Yh AY Tu Chengjian C Shen Shichen S Xu Ding D Oursler Merry J MJ Qu Jun J Yang Shuying S

JBMR plus 20180707 6

Osteoclasts are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). <i>In vitro</i> models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly differ ...[more]

PMID: 30460336

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Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.

			Action	DRS
	BMM_0d_1.msf	Msf
	BMM_0d_1.raw	Raw
	BMM_0d_2.msf	Msf
	BMM_0d_2.raw	Raw
	BMM_0d_3.msf	Msf