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Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis
Author(s) -
Li Yuyu,
Shi Zhenqi,
Jules Joel,
Chen Shenyuan,
Kesterson Robert A,
Zhao Dongfeng,
Zhang Ping,
Feng Xu
Publication year - 2019
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.3810
Subject(s) - rankl , osteoclast , rank ligand , microbiology and biotechnology , chemistry , in vitro , activator (genetics) , biology , receptor , biochemistry
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: PVQEET 560–565 (Motif 2) and PVQEQG 604–609 (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 (RANK KI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANK KI/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 RANK KI/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 RANK KI/KI BMMs, but it cannot stimulate c‐Fos or NFATc1 in the RANK KI/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 RANK KI/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.