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The receptor activator of nuclear factor (NF)-κB 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-κB (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 1α,25-dihydroxyvitamin D3 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.

Determination of Three Isoforms of the Receptor Activator of Nuclear Factor-κΒ Ligand and Their Differential Expression in Bone and Thymus*