Cloning, sequencing, and functional characterization of the rat homologue of receptor activator of NF-kappa B ligand

A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF-kappaB ligand/osteoprotegerin ligand/osteoclast differentiation fact or/tumor necrosis factor (TNF)-related activation-induced cytokine (RANKL/O PGL/ODF/TRANCE) was cloned and sequenced from tibias of ovariectomized (OVX ) rats. The predicted amino acid sequence of rat RANKL (rRANKL) has 84% and 96% identity to that of human and mouse RANKL, respectively, and 35% and 3 7% similarity to that of human and mouse TNF-related apoptosis-inducing lig and (TRAIL), respectively. RANKL transcripts were expressed abundantly in t he thymus and bone tissues of OVX rats. rRANKL has a single hydrophobic reg ion between residues 53 and 69, which is most likely to serve as a transmem brane domain, The long C-terminal region containing beta -sheet-forming seq uences of the TNF-like core is considered the extracellular region. Three t runcated domains within the TNF-like core region were expressed as glutathi one S-transferase (GST) fusion proteins and investigated for their ability to induce osteoclastogenesis, The results showed that GST-rRANKL, (aa160-31 8) containing the full TNF-like core region had the highest capability to i nduce the formation of osteoclast-like cells from RAW(264.7) cells, GST-rRA NKL (aa239-318 and aa160-268) had lesser degrees of osteoclast inductivity, Furthermore, the GST-rRANKL (aa160-318) is capable of (1) inducing osteocl ast formation from rat spleen cells in the presence of macrophage colony-st imulating factor (M-CSF), (2) stimulating mature rat osteoclast polarizatio n and bone resorption ex vivo, and (3) inducing systemic hypercalcemia in v ivo; thus the full TNF-like core region of rRANKL is an important regulator of calcium homeostasis and osteoclastic function.