Determination of three isoforms of the receptor activator of nuclear factor-kappa B ligand and their differential expression in bone and thymus

The receptor activator of nuclear factor (NF)-kappaB ligand [RANKL; also kn own as tumor necrosis factor-related activation-induced cytokine, osteoprot egerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-kappaB (RANK) and act not only as a key facto r for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL, 2 has a s horter 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-E 1, all three RANKL isoforms were detected, but the expression of RANKL 2 wa s preferentially suppressed by treatment with 1 alpha ,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 ce lls 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 thre e isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with ea ch 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 RANXL- RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentia tion than previously thought.