Abstract
Osteoclasts are derived from hematopoietic stem cells. Monocyte preosteoclasts obtain resorbing activity via cell–cell fusion to generate multinucleated cells. However, the mechanisms and molecules involved in the fusion process are poorly understood. In this study, we performed RNA sequencing with single nucleated cells (SNCs) and multinucleated cells (MNCs) to identify the fusion-specific genes. The SNCs and MNCs were isolated under the same conditions during osteo- clastogenesis with receptor activator of the nuclear factor-κB ligand (RANKL) administration. Based on this analysis, the expression of seven genes was found to be significantly increased in MNCs, but decreased in SNCs, compared to that in bone marrow-derived macrophages. We then generated knockout macrophage cell lines using a CRISPR-Cas9 genome-editing tool to examine their function during osteoclastogenesis. Interestingly, Calcrl-, Marco-, or Ube3a-deficient cells could not develop multinucleated giant osteoclasts upon RANKL stimulation. However, Tmem26-deficient cells fused more efficiently than control cells. Our findings demonstrate that Calcrl, Marco, and Ube3a are novel determinants of osteoclastogenesis, especially with respect to cell fusion, and highlight po- tential targets for osteoporosis therapy.