A missense mutation sheds light on a novel structure–function relationship of RANKL

Abstract

The tumor necrosis factor (TNF)‐like core domain of receptor activator of nuclear factor‐κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast‐poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF‐like core domain. However, the structure–function relationship of this mutation is not clear. Sequence‐based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site‐directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast‐related signal transduction. However, no antagonistic effect was found in M200s against wild‐type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor–ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199‐induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

CYT387, a JAK-Specific Inhibitor Impedes Osteoclast Activity and Oophorectomy-Induced Osteoporosis via Modulating RANKL and ROS Signaling Pathways

“…To investigate whether CYT387 pre-treatment affects the transcription of NFATc1, the luciferase reporter genes were transfected into RAW 264.7 cells as previously described ( Qiu et al, 2021 ). The transfected cells were subsequently incubated in 48-well plates containing 5 × 10 4 cells/well and stimulated for 24 h using RANKL (50 ng/ml) and increasing doses of CYT387 (0.5, 1 µM).…”

Cycloastragenol Attenuates Osteoclastogenesis and Bone Loss by Targeting RANKL-Induced Nrf2/Keap1/ARE, NF-κB, Calcium, and NFATc1 Pathways

“…RAW264.7 cell lines transfected with NF-κB, NFATc1, and ARE luciferase reporter genes were used following the previous methods ( Qiu et al, 2021 ), and these cell lines were cultured by D-MEM containing 10% FBS, 1% PS. The next day, after being treated with CAG (2.5, 5, 10 μM) for 60 min, the cells were stimulated by RANKL for stated durations, which were 6 h (NF-κB), 24 h (NFATc1), and 48 h (Nrf2/ARE).…”

The Pathophysiology of Osteoporosis after Spinal Cord Injury

“…RANKL is also a member of the TNF superfamily, and demonstrates high conservation amongst species [ 84 , 92 , 93 ]. In humans, the RANKL gene is located on chromosome 13q14, spanning approximately 36 kb of DNA [ 90 ].…”

“…Recent studies have suggested that osteocytes express high levels of RANKL in order to activate osteoclastogenesis. Knockdown models of RANKL expression in osteocytes demonstrate inhibition of osteoclast activity, increased bone density, and severe osteopetrosis [ 41 , 91 , 93 ].…”