RANK IVVY motif plays crucial roles in osteoclastogenesis.

RANKL and its receptor RANK play a vital role in osteoclastogenesis. RANK primarily recruits TRAFs to promote osteoclastogenesis but also contains an TRAF-independent motif (IVVY), which mediates osteoclast lineage commitment in vitro. Here, we have developed knockin mice in which inactivating mutations are introduced in the IVVY motif (IVVY to IVAF). Homozygous knockin (RANK) mice are viable and born at the expected Mendelian ratio. Micro-computed tomography (μCT) and histomorphometric analyses of femurs of wild type (RANK) and RANK mice reveal significant increases in trabecular bone mass in RANK compared to age and sex matched RANK mice due to impaired osteoclastogenesis in vivo. Bone marrow macrophages (BMMs) from RANK mice do not form osteoclasts in vitro upon M-CSF and RANKL treatment. RANKL-induced activation of NF-ĸB, ERK, p38 and JNK pathways in RANK BMMs remains intact, but RANKL-induced expression of c-Fos and NFATc1 is impaired in RANK BMMs. Consistent with the crucial role of the IVVY motif in priming BMMs into the osteoclast lineage, RANKL-primed RANK BMMs do not form osteoclasts in response to subsequent Porphyromonas gingivalis (Pg)-stimulation, indicating that the IVVY Motif plays a role in Pg-induced osteoclastogenesis. Mechanistically, RANK IVVY motif mediates Pg-induced osteoclast gene expression by rendering NFATc1 and c-Fos genes responsive to Pg stimulation. Consistently, cell penetrating peptides fused to RANK segments containing the IVVY motif impair Pg-induced osteoclastogenesis by impairing RANKL-activated c-Fos and NFATc1 expression. In conclusion, the RANK IVVY motif plays crucial roles in osteoclastogenesis in vivo and modulates Pg-mediated osteoclast formation in vitro.