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

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). In vitro 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 different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts 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 osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. 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 discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M-CSF-dependent signaling and functions.