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 RANKL(G278R) 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.
Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a K d of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins.
Receptor activator of NF-kB ligand (RANKL) plays a key role in osteoclast-induced bone resorption across a range of degenerative bone diseases, and its specific inhibition has been recently approved as a treatment for women with postmenopausal osteoporosis at high or increased risk of fracture in the United States and globally. In the present study, we generated transgenic mice (TghuRANKL) carrying the human RANKL (huRANKL) genomic region and achieved a physiologically relevant pattern of RANKL overexpression in order to establish novel genetic models for assessing skeletal and extraskeletal pathologies associated with excessive RANKL and for testing clinical therapeutic candidates that inhibit human RANKL. TghuRANKL mice of both sexes developed early-onset bone loss, and the levels of huRANKL expression were correlated with bone resorption and disease severity. Low copy Tg5516 mice expressing huRANKL at low levels displayed a mild osteoporotic phenotype as shown by trabecular bone loss and reduced biomechanical properties. Notably, overexpression of huRANKL, in the medium copy Tg5519 line, resulted in severe early-onset osteoporosis characterized by lack of trabecular bone, destruction of the growth plate, increased osteoclastogenesis, bone marrow adiposity, increased bone remodeling, and severe cortical bone porosity accompanied by decreased bone strength. An even more severe skeletal phenotype developed in the high copy Tg5520 founder with extensive soft tissue calcification. Model validation was further established by evidence that denosumab, an antibody that inhibits human but not murine RANKL, fully corrected the hyperresorptive and osteoporotic phenotypes of Tg5519 mice. Furthermore, overexpression of huRANKL rescued osteopetrotic phenotypes of RANKL-defective mice. These novel huRANKL transgenic models of osteoporosis represent an important advance for understanding the pathogenesis and treatment of high-turnover bone diseases and other disease states caused by excessive RANKL.
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