Cloning, sequencing, and functional characterization of the rat homologue of receptor activator of NF-kappaB ligand.
A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF-kappaB 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 beta-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.
Xu
J
J
Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Australia.
Tan
J W
JW
Huang
L
L
Gao
X H
XH
Laird
R
R
Liu
D
D
Wysocki
S
S
Zheng
M H
MH
D000595:Amino Acid Sequence; D000818:Animals; D001862:Bone Resorption; D001842:Bone and Bones / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D016764:Cell Polarity / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006801:Humans; D006934:Hypercalcemia / Q000139:chemically induced; D008024:Ligands; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010052:Ovariectomy; D053245:RANK Ligand; D051381:Rats; D017207:Rats, Sprague-Dawley; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D013950:Thymus Gland / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D002352:Carrier Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor