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

SIMPLE SUMMARY: The formation of multinucleated cells is critical for mature osteoclast generation. For this to happen, mononuclear pre-osteoclasts migrate in proximity to other pre-osteoclasts to fuse together. This finely regulated process is dependent on the “feeling” and “recognition” between neighboring cells. In this study, we focused on pre-osteoclast fusion timing and behavior, and highlighted changes in morphological and cytoskeletal organization during 4 days of osteoclast differentiation. For the first time, interesting cellular extensions have been described, with the presumed function to serve as migration mediators. The sub-cellular localization of key proteins correlated with the osteoclast maturation timing. In particular, for the first time, a relationship of mutual exclusion from the nuclei has been shown between two mitogen activated protein kinases and a master transcription factor. The different trends in the expression of some genes involved in the osteoclast fusion process were related to osteoclast differentiation timing. Although further investigation is needed, we are confident that this study will contribute to the understanding of the mechanisms regulating the initial processes of osteoclastogenesis, including migration and fusion, which in turn are of fundamental importance for the management of many bone-related diseases, such as osteoporosis, osteopetrosis and rheumatoid arthritis. 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.