Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of <i>Dennd2c</i> promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, <i>Dennd2c</i> knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of <i>Dennd2c</i> inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. <i>Dennd2c</i>-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of <i>Dennd2c</i>-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of <i>Dennd2c</i>-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.