Rab44, a novel large Rab GTPase, negatively regulates osteoclast differentiation by modulating intracellular calcium levels followed by NFATc1 activation

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca²⁺ transients upon RANKL stimulation, and particularly regulated lysosomal Ca²⁺ influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca²⁺ levels followed by NFATc1 activation.