Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Abstract

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

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“…The methods suggested by Lampiasi, Nadia et. al, 2021 [ 35 ] were applied in our study for osteoclasts differentiation from murine RAW264.7 cells stimulated by RANKL. Western blot analyses showed that compared with negative control (NC) or RAW264.7 cells co-cultured with miR-21 inhibitor-treated AFs, RAW264.7 cells co-cultured with miR-21 mimic pre-treated AFs showed upregulated expression of osteoclast differentiation markers, matrix metallopeptidase (MMP) 9, receptor activator of NFκB ligand (RANKL), triiodothyronine receptor auxiliary protein (TRAP), and osteoprotegerin (OPG) proteins ( Figure 3 B).…”

“…The method suggested by Lampiasi, Nadia et al, 2021 with little modification [ 35 ] was applied in our study for osteoclasts (OC) differentiation from murine RAW 264.7 cells. In the brief to induce OC differentiation, the murine cells were suspended in an alpha-minimal essential medium (α-MEM Gibco, Grand Island) with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), 100-U/mL penicillin, and 100-µg/mL streptomycin; after 24 h of the cultivation period, the previous α-MEM media was replaced with serum-free-α-MEM medium supplemented with 50-ng/mL RANKL-Recombinant-Protein (ThermoFisher, RP-8601) and human M-CSF (20 ng/mL; ThermoFisher, RP-8615).…”

MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation

“…We have previously shown the timing and behavior of pre-OCs differentiation after RANKL stimulation of RAW264.7 cells over a 4-day period and at different levels, i.e., cell morphology, cytoskeletal organization, protein distribution, signaling pathways role, and OC-specific gene expression [ 4 , 5 , 29 ]. In particular, based on our previous studies on the expression of NFATc1 showing that both mRNA and protein levels increased already on the first day of RANKL stimulation, we established that this was the best RANKL stimulation time to perform miRNA expression analyses.…”

“…Focal adhesion (04510, within “cellular processes”) and ECM–receptor interaction (04512, within “signal transduction”) are pathways that seem very interesting to analyze, partly because we have recently evaluated the differentiation of OCs on different substrates [ 5 ]. Focal adhesion pathway was significantly associated with miRNAs belonging to all the groups ( Table 3 ), while ECM–receptor interaction was associated only with miRNAs of the group 3 ( Table 3 ).…”

“…NFATc1 is the master regulator of osteoclastogenesis, and its expression and activation are tightly regulated at several levels [ 2 , 3 ]. In undifferentiated cells (not supplied with RANKL), NFATc1 is poorly expressed [ 4 ], heavily phosphorylated [ 1 ], and located in the cytoplasm [ 5 ]. Pre-OCs exposure to RANKL triggers an increase in free intracellular Ca 2+ levels, which signals calmodulin (CALM), a calcium binding protein, to activate the Ca 2+ -dependent phosphatase Calcineurin (CN).…”