title: OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation.
abstract: The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.
authors.affiliation: INSERM, ERI 7, Nantes F-44035, France.
authors.forename: Steeve Kwan
authors.initials: SK
authors.lastname: Tat
authors.forename: Marc
authors.initials: M
authors.lastname: Padrines
authors.forename: Sandrine
authors.initials: S
authors.lastname: Theoleyre
authors.forename: Severine
authors.initials: S
authors.lastname: Couillaud-Battaglia
authors.forename: Dominique
authors.initials: D
authors.lastname: Heymann
authors.forename: Françoise
authors.initials: F
authors.lastname: Redini
authors.forename: Yannick
authors.initials: Y
authors.lastname: Fortun
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism* / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053244:Osteoprotegerin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000187:drug effects; D053245:RANK Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D053667:Syndecans / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D002966:Clathrin; D015853:Cysteine Proteinase Inhibitors; D053244:Osteoprotegerin; D061988:Proteasome Inhibitors; D053245:RANK Ligand; D053667:Syndecans; C067713:lactacystin; D002738:Chloroquine; D006493:Heparin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine