Upon receptor activator of NF‐κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor‐associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560–565 (Motif 2) and PVQEQG604–609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock‐in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age‐ and sex‐matched heterozygous KI (RANK+/KI) and wild‐type (RANK+/+) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony‐stimulating factor (M‐CSF) and RANKL in vitro. RANKL is able to activate the NF‐κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c‐Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)‐mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL‐primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg‐induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg‐induced osteoclastogenesis by stimulating c‐Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c‐Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell‐penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL‐ and Pg‐mediated osteoclastogenesis. The CPP conjugates abrogate RANKL‐stimulated c‐Fos and NFATc1 expression but do not affect RANKL‐induced activation of NF‐κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg‐induced osteoclastogenesis in vitro.
Keywords: OSTEOCLASTOGENESIS; RANKL; RANK SIGNALING; PORPHYROMONAS GINGIVALIS