Determination of three isoforms of the receptor activator of nuclear factor-kappaB ligand and their differential expression in bone and thymus.
The receptor activator of nuclear factor (NF)-kappaB ligand [RANKL; also known as tumor necrosis factor-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-kappaB (RANK) and act not only as a key factor for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL 2 has a shorter intracellular domain than the original RANKL (RANKL 1), and RANKL 3 lacks a transmembrane domain and was thought to act as a soluble form. In the bone marrow stromal cell line ST2 and preosteoblastic cell line MC3T3-E1, all three RANKL isoforms were detected, but the expression of RANKL 2 was preferentially suppressed by treatment with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone. In young adult thymus, CD4(-)CD8(-) double-negative cells were positive for all three isoforms, CD4(+)CD8(+) double-positive cells were positive for RANKL 1 and RANKL 3 but negative for RANKL 2, and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive cells were positive for all three isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with each RANKL isoform indicated that the three RANKL isoforms were translated, and RANKL 2 protein predominantly stayed in the endoplasmic reticulum and Golgi networks. These results indicate that there are three kinds of RANKL-RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentiation than previously thought.
Department of Pathology and Immunology, Aging and Developmental Science, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan. toru.pth2@med.tmd.ac.jp
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Ikeda
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Kasai
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Utsuyama
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Hirokawa
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D007536:Isomerism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000502:physiology; D053245:RANK Ligand; D051381:Rats; D053246:Receptor Activator of Nuclear Factor-kappa B; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018124:Receptors, Tumor Necrosis Factor; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha