A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF

• Published in: Human molecular genetics 2012-02, Vol.21 (4), p.784-798
• Main Authors: Douni, Eleni, Rinotas, Vagelis, Makrinou, Eleni, Zwerina, Jochen, Penninger, Josef M., Eliopoulos, Elias, Schett, Georg, Kollias, George
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Amino acids; Animal models; Animals; Biological and medical sciences; Bone diseases; Bone resorption; Codons; Disease Models, Animal; Diseases of the osteoarticular system; Ethyl nitrosourea; Ethylnitrosourea; Fundamental and applied biological sciences. Psychology; Genes, Dominant - genetics; Genetics of eukaryotes. Biological and molecular evolution; Hydrophobicity; Malformations and congenital and or hereditary diseases involving bones. Joint deformations; Medical sciences; Mice; Molecular and cellular biology; Monomers; Mutation; Mutation, Missense - genetics; Osteoclasts; Osteoclasts - cytology; Osteoclasts - metabolism; Osteopetrosis; Osteopetrosis - chemically induced; Osteopetrosis - genetics; Osteoporosis; Point Mutation - genetics; Protein Binding; Protein Multimerization - genetics; random mutagenesis; RANK Ligand - antagonists & inhibitors; RANK Ligand - chemistry; RANK Ligand - genetics; RANK Ligand - metabolism; Receptor Activator of Nuclear Factor-kappa B - metabolism; Receptor mechanisms; TRANCE protein; Tumor necrosis factor; Tumor necrosis factor receptors; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/ddr510

Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumor necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis (ARO), whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach using N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we generated a novel mouse model of ARO caused by a new loss-of-function allele of Rankl with a glycine-to-arginine mutation at codon 278 (G278R) at the extracellular inner hydrophobic F β-strand of RANKL. Mutant mice develop severe osteopetrosis similar to Rankl-deficient mice, whereas exogenous administration of recombinant RANKL restores osteoclast formation in vivo. We show that RANKLG278R monomers fail to assemble into homotrimers, are unable to bind and activate the RANK receptor and interact with wild-type RANKL exerting a dominant-negative effect on its trimerization and function in vitro. Since G278 is highly conserved within the TNF superfamily, we identified that a similar substitution in TNF, G122R, also abrogated trimerization, binding to TNF receptor and consequently impaired TNF biological activity. Notably, SPD304, a potent small-molecule inhibitor of TNF trimerization that interacts with G122, also inhibited RANKL activity, suggesting analogous inhibitory mechanisms. Our results provide a new disease model for ARO and identify a functional amino acid in the TNF-like core domain essential for trimer formation both in RANKL and in TNF that could be considered a novel potential target for inhibiting their biological activities.