CCAR2 reduces the number of osteoclasts by controlling osteoclast apoptosis

Estrogen-associated signals regulating osteoclast apoptosis control osteoclast numbers. Enhancing anti-apoptotic activity during M-CSF and RANKL stimulation, which promotes osteoclast differentiation, increases the number of osteoclasts. Cell cycle and apoptosis regulator 2 (CCAR2) significantly modulates cancer cell survival and apoptosis by interacting with estrogen receptor α (ERα). Because CCAR2 regulates cell fate through ERα, we hypothesize that CCAR2 may inhibit the formation of bone-resorbing osteoclasts. We screen a public database of inbred mouse strain information to identify correlations between Ccar2 mRNA expression in bone and bone mineral density (BMD). We find that Ccar2 expression correlated positively with femoral and spine BMD. In addition, an increased abundance of osteoclasts is observed in aged mice, but the expression of CCAR2 in osteoclasts is lower in old mice than in young mice. To identify the role of CCAR2 in osteoclasts, we generate CCAR2 knockout (KO) RAW 264.7 cells using CRISPR-Cas9. The CCAR2-KO cells exhibit increased osteoclast numbers, reduced intracellular ROS levels, and decreased apoptosis during M-CSF- and RANKL-induced osteoclastogenesis. Mechanistically, CCAR2 binds ERα, altering its nuclear translocation and increasing apoptotic transcriptional activity. These findings suggest that CCAR2 inhibits osteoclast numbers by increasing ERα-mediated apoptosis.