Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF‐κB Ligand

A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF‐κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/tumor necrosis factor (TNF)‐related activation‐induced cytokine (RANKL/OPGL/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 37% similarity to that of human and mouse TNF‐related apoptosis‐inducing ligand (TRAIL), respectively. RANKL transcripts were expressed abundantly in the thymus and bone tissues of OVX rats. rRANKL has a single hydrophobic region between residues 53 and 69, which is most likely to serve as a transmembrane domain. The long C‐terminal region containing β‐sheet‐forming sequences of the TNF‐like core is considered the extracellular region. Three truncated domains within the TNF‐like core region were expressed as glutathione S‐transferase (GST) fusion proteins and investigated for their ability to induce osteoclastogenesis. The results showed that GST‐rRANKL (aa160‐318) containing the full TNF‐like core region had the highest capability to induce the formation of osteoclast‐like cells from RAW264.7 cells. GST‐rRANKL (aa239‐318 and aa160‐268) had lesser degrees of osteoclast inductivity. Furthermore, the GST‐rRANKL (aa160‐318) is capable of (1) inducing osteoclast formation from rat spleen cells in the presence of macrophage colony‐stimulating factor (M‐CSF), (2) stimulating mature rat osteoclast polarization and bone resorption ex vivo, and (3) inducing systemic hypercalcemia in vivo; thus the full TNF‐like core region of rRANKL is an important regulator of calcium homeostasis and osteoclastic function.