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^535-538), 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^AF/AF) 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^AF/AF mice reveal significant increases in trabecular bone mass in RANK^AF/AF compared to age and sex matched RANK^+/+ mice due to impaired osteoclastogenesis in vivo. Bone marrow macrophages (BMMs) from RANK^AF/AF 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^AF/AF BMMs remains intact, but RANKL-induced expression of c-Fos and NFATc1 is impaired in RANK^AF/AF BMMs. Consistent with the crucial role of the IVVY motif in priming BMMs into the osteoclast lineage, RANKL-primed RANK^AF/AF 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.