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

Authors

  • Jiake Xu,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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  • Jamie Wy Tan,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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    • These authors contributed equally to this work.

  • Lin Huang,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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    • These authors contributed equally to this work.

  • Xiu-Hui Gao,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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  • Rebecca Laird,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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  • Dan Liu,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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  • Stan Wysocki,

    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
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  • Ming H. Zheng M.D, Ph.D., M.R.C.Path.

    Corresponding author
    1. Department of Orthopedic Surgery, The University of Western Australia, WA Institute for Medical Research, QEII Medical Center, Nedlands, Western Australia, Australia
    • Department of Orthopedic Surgery, University of Western Australia, Nedlands 6009 WA, Australia
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  • The sequence data has been submitted to the GenBank databases under access number AF187319.

Abstract

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.

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